Hydrophobically-modified protein compositions and methods

ABSTRACT

Hydrophobically-modified proteins and methods of making them are described. A hydrophobic moiety is attached to a surface amino acid residue of the protein. The hydrophobic moiety can be a lipid or a peptide. Alternatively, the protein can be derivatized by a wide variety of chemical reactions that append a hydrophobic structure to the protein. The preferred protein is of mammalian origin and is selected from the group consisting of Sonic, Indian, and Desert hedgehog. The hydrophobic moiety is used as a convenient tether to which may be attached a vesicle such as a cell membrane, liposome, or micelle.

BACKGROUND OF THE INVENTION

It is known that certain proteins exhibit greater biological activitywhen attached to other moieties, either by formation of multimericcomplexes, where the proteins have an opportunity to act in concert, orthrough other alterations in the protein's physico-chemical properties,such as the protein's absorption, biodistribution and half life. Thus,one current area of research in biotechnology involves the developmentof methods to modify the physico-chemical properties of proteins so thatthey can be administered in smaller amounts, with fewer side effects, bynew routes, and with less expense.

For example, the binding affinity of any single protein (such as aligand for its cognate receptor) may be low. However, cells normallyexpress hundreds to thousands of copies of a particular surfacereceptor, and many receptor-ligand interactions take placesimultaneously. When many surface molecules become involved in binding,the total effective affinity is greater than the sum of the bindingaffinities of the individual molecules. By contrast, when ligandproteins are removed from the cell surface and purified, or isolated byrecombinant DNA techniques for use, e.g., as therapeutics, they act asmonomers and lose the advantage of acting in concert with many othercopies of the same protein associated closely on the surface of a cell.Thus isolated, the low affinity of a protein for its receptor may becomea serious drawback to its effectiveness as a therapeutic to block aparticular binding pathway, since it must compete against the highavidity cell-cell interactions. Effective treatment might requireconstant administration and/or high doses. Such drawbacks might beavoided, however, if a means could be found to provide multimeric formsof an isolated protein.

Similarly, it would be useful to modify other physico-chemicalproperties of biologically active proteins so that, for instance, aprotein is induced to associate with a membrane thus localizing it atthe site of administration and enhancing its ability to bind to, orotherwise interact with, a particular target. Such changes may alsoaffect the pharmaco-distribution of the protein.

Several methods of generating coupled proteins have been developed. Manyof these methods are not highly specific, i.e., they do not direct thepoint of coupling to any particular site on the protein. As a result,conventional coupling agents may attack functional sites or stericallyblock active sites, rendering the coupled proteins inactive.Furthermore, the coupled products may be oriented so that the activesites cannot act synergistically, thereby rendering the products no moreeffective than the monomeric protein alone.

As an additional motivation to find new methods for proteinmodification, proteins with an N-terminal cysteine residue aresusceptible to oxidation or other chemical modifications that maycompromise activity or half-life. Additionally, certain buffers commonlyused in protein purification have components or impurities that canmodify the N-terminal cysteine. Even when these buffers are avoided, theN-terminal cysteine is modified over time, perhaps due to chemicals inthe storage vials or in the air. Consequently, formulation buffers mustinclude a protective agent, such as dithiothreitol, to prevent cysteinemodification and/or oxidation. However, protective agents havesignificant biological activity of their own and they may thereforecomplicate experiments and adversely affect the therapeutic utility of aformulation.

Accordingly, there is a need in the art to develop more specific meansto obtain derivatized products or multimeric forms thereof so as toalter the properties of the protein in order to affect its stability,potency, pharmacokinetics, and pharmacodynamics.

SUMMARY OF THE INVENTION

In one aspect of the invention, we have solved the problem of finding away to conveniently make modified forms of biologically active proteins.Methods of the invention can be used to derive multimeric forms of theproteins and/or can be used to change their physico-chemical properties.We have found that modifying a protein (i.e, adding or appending ahydrophobic moiety to an existing amino acid or substituting ahydrophobic moiety for an amino acid) so as to introduce the hydrophobicmoiety onto a protein can increase the protein's biological activityand/or its stability. For example, an N-terminal cysteine can be used asa convenient “target” to attach a hydrophobic moiety (e.g., a lipid) andthereby modify biologically active proteins.

Alternatively, a hydrophobic moiety can be attached to a C-terminalresidue of a biologically active protein, such as hedgehog protein, tomodify the protein's activity. A hydrophobic moiety can also be appendedto an internal amino acid residue to enhance the protein's activity,provided the modification does not affect the activity of the protein,e.g., the proteins ability to bind to a receptor or co-receptor, oraffect the protein's 3-dimensional structure. Preferably, thehydrophobic moiety is appended to an internal amino acid residue that ison the surface of the protein when the protein is in its native form.The hydrophobic modification of the invention provides a genericallyuseful method of creating proteins with altered physico-chemicalproperties as compared to non-modified forms.

This invention originated from the discovery that when we expressedfull-length Sonic hedgehog protein in insect and in mammalian cells, themature form of the protein (residues 1-174 in the mature sequence), inaddition to having cholesterol at the C-terminus, is also derivatized atits N-terminal end with a fatty acid. Significantly, this form ofhedgehog exhibited about a 30-fold increase in potency as compared tosoluble, unmodified hedgehog in an in vitro assay.

One aspect of the invention is therefore an isolated, protein comprisingan N-terminal amino acid and a C-terminal amino acid, wherein theprotein is selected from the group consisting of a protein with anN-terminal cysteine that is appended with at least one hydrophobicmoiety; a protein with an N-terminal amino acid that is not a cysteineappended with a hydrophobic moiety; and a protein with a hydrophobicmoiety substituted for the N-terminal amino acid. The hydrophobic moietycan be a hydrophobic peptide or any lipid or any other chemical moietythat is hydrophobic.

The protein may be modified at its N-terminal amino acid and preferablythe N-terminal amino acid is a cysteine or a functional derivativethereof. The protein may be modifed at its C-terminal amino acid or atboth the N-terminal amino acid and the C-terminal amino acid, or at atleast one amino acid internal to (i.e., intermediate between) theN-terminal and C-terminal amino acids, or various combinations of theseconfigurations. The protein can be an extracellular signaling proteinand in preferred embodiments, the protein is a hedgehog proteinobtainable from a vertebrate source, most preferably obtainable from ahuman and includes Sonic, Indian, and Desert hedgehog.

Another embodiment is an isolated, protein of the form: A-Cys-[Sp]-B-X,wherein A is a hydrophobic moiety;

-   -   Cys is a cysteine or functional equivalent thereof;    -   [Sp] is an optional spacer peptide sequence;    -   B is a protein comprising a plurality of amino acids, including        at least one optional spacer peptide sequence; and    -   X is optionally another hydrophobic moiety linked to the        protein.

The isolated protein can be an extracellular signaling protein,preferably a hedgehog protein. This protein can be modified at at leastone other amino acid with at least one hydrophobic moiety. In otherembodiments, the protein is in contact with a vesicle in selected fromthe group consisting of a cell membrane, micelle and liposome.

Another aspect of the invention is an isolated, protein having aC-terminal amino acid and an N-terminal thiaproline group, thethiaproline group formed by reacting an aldehyde with an N-terminalcysteine of the protein. A further aspect of the invention is isolated,protein having a C-terminal amino acid and an N-terminal amide group,the amide group formed by reacting a fatty acid thioester with anN-terminal cysteine of the protein. Yet another aspect of the inventionis an isolated, protein having a C-terminal amino acid and an N-terminalmaleimide group, the N-terminal maleimide group formed by reacting amaleimide group with the N-terminal cysteine of the protein. Yet anotheraspect of the invention is an isolated, protein having a C-terminalamino acid and an N-terminal acetamide group. A further aspect of theinvention is an isolated, protein having a C-terminal amino acid and anN-terminal thiomorpholine group.

In these embodiments, the C-terminal amino acid of the protein can bemodified with an hydrophobic moiety. The isolated protein can be anextracellular signaling protein, most preferably a hedgehog protein.

Methods of the invention include a method of generating a multivalentprotein complex comprising the step of linking, in the presence of avesicle, a hydrophobic moiety to an N-terminal cysteine of a protein, ora functional equivalent of the N-terminal cysteine. The linking step mayinclude linking a lipid moiety which is selected from saturated andunsaturated fatty acids having between 2 and 24 carbon atoms. Theprotein can be an extracellular signaling protein, preferably a hedgehogprotein selected from the group consisting of Sonic, Indian and Deserthedgehog.

Yet another method of the invention is a method for modifying aphysico-chemical property of a protein, comprising introducing at leastone hydrophobic moiety to an N-terminal cysteine of the protein or to afunctional equivalent of the N-terminal cysteine. The hydrophobic moietycan be a lipid moiety selected from saturated and unsaturated fattyacids having between 2 and 24 carbon atoms. It can also be a hydrophobicprotein The protein modified using this method can be an extracellularsignaling protein, preferably a hedgehog protein selected from the groupconsisting of Sonic, Indian and Desert hedgehog. A protein complex,produced by these methods are also encompassed by the present invention.

Other extracellular signaling proteins besides hedgehog includegelsolin; an interferon, an interleukin, tumor necrosis factor, monocytecolony stimulating factor, granulocyte colony stimulating factor,granulocyte macrophage colony stimulating factor, erythropoietin,platelet derived growth factor, growth hormone and insulin.

Another method is a method for modifying a protein (such as anextracellular signaling protein) that has an N-terminal cysteine. Thismethod comprises reacting the N-terminal cysteine with a fatty acidthioester to form an amide, wherein such modification enhances theprotein's biological activity.

Yet another method is a method for modifying a protein (such as anextracellular signaling protein) having an N-terminal cysteine, whichcomprising reacting the N-terminal cysteine with a maleimide group,wherein such modification enhances the protein's biological activity.Other embodiments of this method involve reacting the N-terminalcysteine with either an aldehyde group, an acetamide group or athiomorpholine group.

A further method is a method for modifying protein (such as anextracellular signaling protein) comprising appending an hydrophobicpeptide to the protein. The hydrophobic moiety can be appended to anamino acid of the protein selected from the group consisting of theN-terminal amino acid, the C-terminal amino acid, an amino acidintermediate between the N-terminal amino acid and the C-terminal aminoacid, and combinations of the foregoing. In one embodiment, the presentinvention provides hedgehog polypeptides which are modified withlipophilic moieties. In certain embodiments, the hedgehog proteins ofthe present invention are modified by a lipophilic moiety or moieties atone or more intenal sites of the mature, processed extracellular domain,and may or may not be also derivatized with lipophilic moieties at the Nor C-terminal residues of the mature polypeptide. In other embodiments,the polypeptide is modified at the C-terminal residue with a hydrophobicmoiety other than a sterol. In still other embodiments, the polypeptideis modified at the N-terminal residue with a cyclic (preferablypolycyclic) lipophilic group. Various combinations of the above are alsocontemplated. A therapeutic method of the invention is a method fortreating a neurological disorder in a patient comprising administeringto the patient a hydrophobically-modified protein of the invention.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. Characterization of a palmitoylated form of Shh. A tethered formof human Shh was immunoaffinity purified from High Five™ insect cellsand analyzed by SDS-PAGE. The protein was stained with Coomassie blue(lane a, Life Technologies, Inc. prestained high molecular weightmarkers; lane b, soluble Shh (0.6 μg); lane c, tethered Shh (0.6 μg);lane d, mixture of soluble plus tethered Shh (0.6 μg each)). The abilityof Shh and Ihh (see lane h) to be modified with palmitic acid wasassayed using a cell-free system described in Example 2. Soluble formsof hedgehog protein (3 μg/sample) were incubated for 1 h with rat livermicrosomes, ATP, CoenzymeA, and ³H-palmitic acid, and then analyzed forpalmitoylation by SDS-PAGE. The samples shown in lanes e-i werevisualized by fluorography (lane e, Shh; lane f, des 1-10 Shh; lane g,Cys-1 to Ser Shh; lane h, Ihh; lane i, His-tagged Shh) and in lanes j-kby Coomassie staining (lane j, Shh; lane k des 1-10 Shh).

FIG. 2. Analysis of purified Shh by ESI-MS. Soluble human Shh (A) andtethered human Shh (B) were analyzed by ESI-MS on a Micromass Quattro IItriple quadrupole mass spectrometer, equipped with an electrospray ionsource. All electrospray mass spectral data were acquired and stored inprofile mode and were processed using the Micromass MassLynx datasystem. Molecular mass spectra are shown (mass assignments weregenerated by the data system).

FIG. 3. Analysis of tethered Shh by reverse phase HPLC. Soluble humanShh (A), tethered human Shh from High Five™ insect cells (B), tetheredhuman Shh from EBNA-293 cells (C), and cell-associated rat Shh (D) weresubjected to reverse phase HPLC on a narrow bore Vydac C₄ column (2.1 mminternal diameter×250 mm). The column was developed with a 30 min, 0-80%acetonitrile gradient in 0.1% trifluoroacetic acid at 0.25 mL/min andthe effluent monitored using a photodiode array detector from 200-300 nm(data shown at 214 nm). Peak fractions were collected and characterizedfurther by SDS-PAGE and MS (data summarized in Tables 3, 4, and 5).

FIG. 4. Characterization of Shh by LC-MS. Tethered human Shh (A) andsoluble human Shh (B) were alkylated with 4-vinylpyridine (1 μL/100 μLsample in 6 M guanidine HCl, 1 mM EDTA, 100 mM Tris HCl pH 8.0), ethanolprecipitated, and digested with endoproteinase Lys-C in 50 mM Tris HClpH 7.0, 2 M urea at an enzyme:protein ratio of 1:5 as describedpreviously (27). The digests were analyzed by reverse phase HPLC in linewith an electrospray Micromass Quattro II triple quadrupole massspectrometer. Scans were acquired throughout the run and processed usingthe Micromass MassLynx data system (total ion chromatograms from theruns are shown). Asterisks indicate the positions of the N-terminalpeptide which were verified either by MALDI PSD or N-terminal Edmansequencing.

FIG. 5. Sequencing of the N-terminal Shh peptide by MALDI PSDmeasurement. The N-terminal endoproteinase Lys-C peptide from tetheredhuman Shh was subjected to MALDI PSD measurement on a Voyager-DE™ STRtime of flight mass spectrometer. The predicted fragmentation patternand nomenclature for the detected fragment ions are shown at the top ofthe panel (PA, palmitoyl acid; 4vp, 4-pyridylethyl group). The remainderof the Figure shows the molecular mass spectrum produced by the run.Relevant ions are denoted using the nomenclature defined in theschematic. Calculated masses (Da) for b₁-b₈ are 447.3, 504.3, 601.4,658.4, 814.5, 871.5, 1018.6, and 1075.6, respectively. For y₁-y₈, themasses (Da) are 147.1, 204.1, 351.2, 408.2, 564.3, 621.3, 718.4, and775.4, respectively. The calculated mass for z₈ is 758.4 Da. Theobserved mass for b₈ contains an additional 18 Da due to an added water.

FIG. 6. Increased activity of tethered Shh in the C3H10T1/2 assay. Therelative potencies of soluble and tethered human Shh alone (A) or in thepresence of the anti-hedgehog neutralizing Mab 5E1 (B) were assessed onC3H10T1/2 cells measuring alkaline phosphatase induction. The numberspresented reflect the averages of duplicate determinations. (A) Serial2-fold dilutions of soluble (6) and tethered (8) Shh were incubated withthe cells for 5 days and the resulting levels of alkaline phosphataseactivity measured at 405 nm using the alkaline phosphatase chromogenicsubstrate p-nitrophenyl phosphate. (B) Serial dilutions of Mab 5E1 wereincubated with soluble Shh (5 μg/mL: black bars) or tethered Shh (0.25μg/mL: gray bars) or vehicle control without Shh added (white bar) for30 min and then subjected to analysis in the C3HT101/2 assay.

FIG. 7. Analysis of Shh in a receptor binding assay. The relativepotency of soluble (6) and tethered (8) Shh for binding to patched wasassessed on patched-transfected EBNA-293 cells by FACS analysis. Serialdilutions of the test samples were incubated with the EBNA-293 cells,washed, and then the percent binding measured by the ability of thesamples to compete with Shh-Ig for binding to the cells. Bound Shh-Igwas quantified by mean fluorescence using a FITC-labeled anti-Igantibody probe as the readout. The data were fitted to a hyperboliccurve by non-linear regression.

FIG. 8. Alignment of N-terminal fragment of human hedgehog proteins. The20 kDa human hedgehog proteins (Sonic “Shh”, Desert “Dhh” and Indian“Ihh”) are aligned with respect to their N-terminal cysteine (Cys-1 inthe mature sequence). This cysteine is normally Cys-24 in thefull-length Shh precursor protein due to the presence of the naturalsignal sequence that is removed during secretion. The actual position ofthe cysteine may vary slightly due to species differences.

FIG. 9. Consensus Sequence of the N-terminal fragment of human hedgehogproteins.

FIG. 10. Effect of lipid chain length on activity of human Sonichedgehog. A series of fatty acid-modified hedgehog proteins wassynthesized according to the present invention and the effect of thefatty acid chain length on hedgehog activity was tested using theC3H10T1/2 alkaline phosphatase induction assay described herein. Theresults are plotted as a bar graph.

FIG. 11. C3H10T1/2 assay of palmitoylated, myristyolated, lauroylated,decanoylated, and octanoylated human Sonic hedgehog. Palmitoylated,lauroylated, decanoylated, and octanoylated human Sonic hedgehogformulated in 5 mM Na₂HPO₄ pH 5.5, 150 mM NaCl, 1% octylglucoside, 0.5mM DTT, and myristoylated human Sonic hedgehog, formulated in 1-50 mMNaCl, 0.5 mM DTT, were assayed on C3H10T1/2 cells measuring alkalinephosphatase induction. The numbers represent the mean of duplicatedeterminations. Serial 3-fold dilutions of palmitoylated (◯),myristoylated (●), lauroylated (□), decanoylated (▪), octanoylated (Δ),and unmodified (▴ and x) human Sonic hedgehog were incubated with thecells for 5 days and the resulting levels of alkaline phosphatasemeasured at 405 nm using the chromogenic substrate p-nitrophenylphosphate. The palmitoylated, myristoylated, lauroylated, anddecanoylated proteins were assayed in one experiment with the unmodifiedprotein shown as (▴), while the octanoylated protein was assayed inanother experiment with the unmodified protein shown as (x). The arrowon the y-axis denotes the background level of alkaline phosphatase inthe absence of added hedgehog protein.

FIG. 12. Generic structures of various hydrophobically-modified forms ofhedgehog. (A) Fatty amide derivative where R=a hydrocarbon chain of afatty acid; (B) thiazolidine derivative where R=a hydrocarbon; (C) aminoacid substitution where R=a hydrophobic amino acid side chain; (D)maleimide derivative where R=a hydrocarbon; (E) SH=free thiol onN-terminal cysteine of wild type hedgehog; (F) an iodoacetamidederivative where R₁=a hydrocarbon and R₂=either H or a hydrocarbon; and(G) thiomorpholinyl derivative where R=a hydrocarbon. For allstructures, HH=hedgehog.

FIG. 13. Relative potency of various hydrophobically-modified forms ofhedgehog in the C3H10T1/2 assay. The EC₅₀ (2 μg/ml) of unmodified wildtype human Sonic hedgehog is designated as 1 x. The potency of the otherproteins is expressed as the ratio of the EC₅₀ of wild type proteindivided by the EC₅₀ of the modified protein. Modifications are at theN-terminus of the protein unless designated otherwise.

FIG. 14. Relative potency of the unmodified, myristoylated, and C1 IImutant of human Sonic hedgehog in a malonate-induced rat striatal lesionassay. The figure shows the reduction in malonate-induced lesion volumewhich results from the administation of either unmodified,myristoylated, or the C1 II mutant of human Sonic hedegehog to the ratstriatum.

FIG. 15. illustrates the specific activities of maleimide modified andunmodified hedgehog polypeptides.

DETAILED DESCRIPTION OF THE INVENTION

This invention is based, in part, on the discovery that human Sonichedgehog, expressed as a full-length construct in either insect or inmammalian cells, has a hydrophobic palmitoyl group appended to theα-amine of the N-terminal cysteine. This is the first example, of whichthe inventors are aware, of an extracellular signaling protein beingmodified in such a manner, and, in contrast to thiol-linked palmiticacid modifications whose attachment is readily reversible, this novelN-linked palmitoyl moiety is likely to be very stable by analogy withmyristic acid modification.

As a direct consequence of this initial discovery, the inventors havefound that increasing the hydrophobic nature of a signaling protein canincrease the protein's biological activity. In particular, the inventorshave found that appending a hydrophobic moiety to a signaling protein,such as a hedgehog protein, can enhance the protein's activity. Theinventors have found that the N-terminal cysteine of biologically activeproteins not only provides a convenient site for appending a hydrophobicmoeity, and thereby modifying the physico-chemical properties of theprotein, but modifications to the N-terminal cysteine can also increasethe protein's stability. Additionally, addition of a hydrophobic moietyto an internal amino acid residue on the surface of the proteinstructure enhances the protein's activity. We use as an example, ourdiscovery of hydrophobic (e.g., lipids and hydrophobic amino acid)modifications of hedgehog protein.

One aspect of the present application is directed to the discovery that,in addition to those effects seen by cholesterol-addition to theC-terminus of extracellular fragments of the protein, at least certainof the biological activities of the hedgehog gene products areunexpectedly potentiated by derivativation of the protein withlipophilic moieties at other sites on the protein and/or by moietiesother than cholesterol. Certain aspects of the invention are directed topreparations of hedgehog polypeptides which are modified at sites otherthan N-terminal or C-terminal residues of the natural processed form ofthe protein, and/or which are modified at such terminal residues withlipophilic moieties other than a sterol at the C-terminus or fatty acidat the N-terminus.

As described in PCT publications WO 95/18856 and WO 96/17924 (all ofwhich are expressly incorporated by reference herein), hedgehogpolypeptides in general are useful in the in vitro and in vivo repairingand/or regulating the functional performance of a wide range of cells,tissues and organs, and have therapeutic uses ragning fromneuroprotection, neuroregeneration, enhancement of neural function,regulation of bone and cartilage formation and repair, regulation ofspermatogenesis, regulation of lung, liver and other organs arising fromthe primative gut, regulation of hematopoietic function, etc.Accordingly, the methods and compositions of the present inventioninclude the use of the derivatized hedgehog polypeptides for all suchuses as hedgehog proteins have been implicated. Moreover, the subjectmethods can be performed on cells which are provided in culture (invitro), or on cells in a whole animal (in vivo).

In one aspect, the present invention provides pharmaceuticalpreparations comprising, as an active ingredient, a hedgehog polypeptidebeing derivatized by one or more lipophilic moieties such as describedherein.

The subject hedgehog treatments are effective on both human and animalsubjects. Animal subjects to which the invention is applicable extend toboth domestic animals and livestock, raised either as pets or forcommercial purposes. Examples are dogs, cats, cattle, horses, sheep,hogs and goats.

The hedgehog proteins are a family of extracellular signaling proteinsthat regulate various aspects of embryonic development both invertebrates and in invertebrates (for reviews see 1,2). The mostwell-characterized hedgehog protein is Sonic hedgehog (Shh), involved inanterior-posterior patterning, formation of an apical ectodermal ridge,hindgut mesoderm, spinal column, distal limb, rib development, and lungdevelopment, and in inducing ventral cell types in the spinal cord,hindbrain and forebrain (3-8). While the mechanism of action of hedgehogproteins is not understood fully, the most recent biochemical andgenetic data suggest that the receptor for Shh is the product of thetumor suppressor gene, patched (9,10) and that other proteins;smoothened (10,11), Cubitus interruptus (12,13), and fused (14) areinvolved in the hedgehog signaling pathway.

Human Shh is synthesized as a 45 kDa precursor protein that is cleavedautocatalytically to yield: (I) a 20 kDa N-terminal fragment that isresponsible for all known hedgehog signaling activity (SEQ ID NOS. 1-4);and (II) a 25 kDa C-terminal fragment that contains the autoprocessingactivity (15-17). The N-terminal fragment consists of amino acidresidues 24-197 of the full-length precursor sequence.

The N-terminal fragment remains membrane-associated through the additionof a cholesterol at its C-terminus (18,19). This cholesterol is criticalfor restricting the tissue localization of the hedgehog signal. Theaddition of the cholesterol is catalyzed by the C-terminal domain duringthe processing step.

All references cited in the detailed description are, unless otherwisestipulated, incorporated herein by reference.

I. Definitions

The invention will now be described with reference to the followingdetailed description of which the following definitions are included:

“amino acid”—a monomeric unit of a peptide, polypeptide, or protein.There are twenty amino acids found in naturally occurring peptides,polypeptides and proteins, all of which are L-isomers. The term alsoincludes analogs of the amino acids and D-isomers of the protein aminoacids and their analogs.

“protein”—any polymer consisting essentially of any of the 20 aminoacids. Although “polypeptide” is often used in reference to relativelylarge polypeptides, and “peptide” is often used in reference to smallpolypeptides, usage of these terms in the art overlaps and is varied.The term “protein” as used herein refers to peptides, proteins andpolypeptides, unless otherwise noted.

“N-terminal end”—refers to the first amino acid (amino acid number 1) ofthe mature form of a protein.

“N-terminal cysteine”—refers to the amino acid residue (number 1) asshown in SEQ ID NOS. 1-4. It also refers to any cysteine at position 1of any other protein, or functional equivalents of this cysteine (SeeSection IV).

“spacer” sequence refers to a short sequence that can be as small as asingle amino acid that may be inserted between an amino acid to behydrophobically modified (such as, for example, the N-terminal cysteineor functional equivalent) and the remainder of the protein. A spacer isdesigned to provide separation between the-hydrophobic modification(e.g., the modified N-terminal cysteine) and the rest of the protein soas to prevent the modification from interfering with protein functionand/or make it easier for the modification (e.g., the N-terminalcysteine) to link with a lipid, vesicle, or other hydrophobic moiety.Thus, if a protein is modified at its N-terminal cysteine and at anamino acid at another site, there may be two, or more, spacer sequences.

“tethered” protein—refers to a hydrophobically-modified proteinaccording to the invention.

“multivalent protein complex”—refers to a plurality of proteins (i.e.,one or more). A lipid or other hydrophobic moiety is attached to atleast one of the plurality of proteins. The lipid or other hydrophobicmoiety may optionally be in contact with a vesicle. If a protein lacks alipid or other hydrophobic moiety, then that protein may be cross-linkedor bind to a protein that does have a lipid or other hydrophobic moiety.Each protein may be the same or different and each lipid or otherhydrophobic moiety may be the same or different.

“vesicle”—refers to any aggregate of lipophilic molecules. The vesiclemay be obtained from a biologic source (e.g., a lipid bilayer such as acell membrane or a cholic acid-derived detergent preparation) or from anon-biologic source (e.g., a non-biologic detergent vesicle as describedin Section VI). The shape, type, and configuration of the vesicle is notintended to limit the scope of this invention.

“functional equivalent” of an amino acid residue (e.g., an N-terminalcysteine)—is (i) an amino acid having similar reactive properties as theamino acid residue that was replaced by the functional equivalent; (ii)an amino acid of a ligand of a polypeptide of the invention, the aminoacid having similar hydrophobic (e.g., lipid) moiety binding propertiesas the amino acid residue that was replaced by the functionalequivalent; (iii) a non-amino acid molecule having similar hydrophobic(e.g., lipid) moiety binding properties as the amino acid residue thatwas replaced by the functional equivalent.

“genetic fusion”—refers to a co-linear, covalent linkage of two or moreproteins or fragments thereof via their individual peptide backbones,through genetic expression of a polynucleotide molecule encoding thoseproteins.

A “chimeric protein” or “fusion protein” is a fusion of a first aminoacid sequence encoding a hedgehog polypeptide with a second amino acidsequence defining a domain foreign to and not substantially homologouswith any domain of hh protein. A chimeric protein may present a foreigndomain which is found (albeit in a different protein) in an organismwhich also expresses the first protein, or it may be an “interspecies”,“intergenic”, etc. fusion of protein structures expressed by differentkinds of organisms. In general, a fusion protein can be represented bythe general formula (X)_(n)-(hh)_(m)-(Y)_(n), wherein hh represents allor a portion of the hedgehog protein, X and Y each independentlyrepresent an amino acid sequences which are not naturally found as apolypeptide chain contiguous with the hedgehog sequence, m is an integergreater than or equal to 1, and each occurrence of n is, independently,0 or an integer greater than or equal to 1 (n and m are preferably nogreater than 5 or 10).

“mutant”—any change in the genetic material of an organism, inparticular any change (i.e., deletion, substitution, addition, oralteration) in a wild type polynucleotide sequence or any change in awild type protein.

“wild type”—the naturally-occurring polynucleotide sequence of an exonof a protein, or a portion thereof, or protein sequence, or portionthereof, respectively, as it normally exists in vivo.

“standard hybridization conditions”—salt and temperature conditionssubstantially equivalent to 0.5×SSC to about 5×SSC and 65° C. for bothhybridization and wash. The term “standard hybridization conditions” asused herein is an operational definition and encompasses a range ofhybridization conditions. See also Current Protocols in MolecularBiology, John Wiley & Sons, Inc. New York, Sections 6.3.1-6.3.6, (1989).

“expression control sequence”—a sequence of polynucleotides thatcontrols and regulates expression of genes when operatively linked tothose genes.

“operatively linked”—a polynucleotide sequence (DNA, RNA) is operativelylinked to an expression control sequence when the expression controlsequence controls and regulates the transcription and translation ofthat polynucleotide sequence. The term “operatively linked” includeshaving an appropriate start signal (e.g., ATG) in front of thepolynucleotide sequence to be expressed, and maintaining the correctreading frame to permit expression of the polynucleotide sequence underthe control of the expression control sequence, and production of thedesired polypeptide encoded by the polynucleotide sequence.

“expression vector”—a polynucleotide, such as a DNA plasmid or phage(among other common examples) which allows expression of at least onegene when the expression vector is introduced into a host cell. Thevector may, or may not, be able to replicate in a cell.

“Isolated” (used interchangeably with “substantially pure”)—when appliedto nucleic acid i.e., polynucleotide sequences that encode polypeptides,means an RNA or DNA polynucleotide, portion of genomic polynucleotide,cDNA or synthetic polynucleotide which, by virtue of its origin ormanipulation: (i) is not associated with all of a polynucleotide withwhich it is associated in nature (e.g., is present in a host cell as anexpression vector, or a portion thereof); or (ii) is linked to a nucleicacid or other chemical moiety other than that to which it is linked innature; or (iii) does not occur in nature. By “isolated” it is furthermeant a polynucleotide sequence that is: (i) amplified in vitro by, forexample, polymerase chain reaction (PCR); (ii) synthesized chemically;(iii) produced recombinantly by cloning; or (iv) purified, as bycleavage and gel separation.

Thus, “substantially pure nucleic acid” is a nucleic acid which is notimmediately contiguous with one or both of the coding sequences withwhich it is normally contiguous in the naturally occurring genome of theorganism from which the nucleic acid is derived. Substantially pure DNAalso includes a recombinant DNA which is part of a hybrid gene encodingadditional hedgehog sequences.

“Isolated” (used interchangeably with “substantially pure”)—when appliedto polypeptides means a polypeptide or a portion thereof which, byvirtue of its origin or manipulation: (i) is present in a host cell asthe expression product of a portion of an expression vector; or (ii) islinked to a protein or other chemical moiety other than that to which itis linked in nature; or (iii) does not occur in nature, for example, aprotein that is chemically manipulated by appending, or adding at leastone hydrophobic moiety to the protein so that the protein is in a formnot found in nature. By “isolated” it is further meant a protein thatis: (i) synthesized chemically; or (ii) expressed in a host cell andpurified away from associated and contaminating proteins. The termgenerally means a polypeptide that has been separated from otherproteins and nucleic acids with which it naturally occurs. Preferably,the polypeptide is also separated from substances such as antibodies orgel matrices (polyacrylamide) which are used to purify it.

“Heterologous promoter”—as used herein is a promoter which is notnaturally associated with a gene or a purified nucleic acid.

“Homologous”—as used herein is synonymous with the term “identity” andrefers to the sequence similarity between two polypeptides, molecules,or between two nucleic acids. When a position in both of the twocompared sequences is occupied by the same base or amino acid monomersubunit (for instance, if a position in each of the two DNA molecules isoccupied by adenine, or a position in each of two polypeptides isoccupied by a lysine), then the respective molecules are homologous atthat position. The percentage homology between two sequences is afunction of the number of matching or homologous positions shared by thetwo sequences divided by the number of positions compared×100. Forinstance, if 6 of 10 of the positions in two sequences are matched orare homologous, then the two sequences are 60% homologous. By way ofexample, the DNA sequences CTGACT and CAGGTT share 50% homology (3 ofthe 6 total positions are matched). Generally, a comparison is made whentwo sequences are aligned to give maximum homology. Such alignment canbe provided using, for instance, the method of Needleman et al., J. MolBiol. 48: 443-453 (1970), implemented conveniently by computer programssuch as the Align program (DNAstar, Inc.). Homologous sequences shareidentical or similar amino acid residues, where similar residues areconservative substitutions for, or “allowed point mutations” of,corresponding amino acid residues in an aligned reference sequence. Inthis regard, a “conservative substitution” of a residue in a referencesequence are those substitutions that are physically or functionallysimilar to the corresponding reference residues, e.g., that have asimilar size, shape, electric charge, chemical properties, including theability to form covalent or hydrogen bonds, or the like. Particularlypreferred conservative substitutions are those fulfilling the criteriadefined for an “accepted point mutation” in Dayhoff et al., 5: Atlas ofProtein Sequence and Structure, 5: Suppl. 3, chapter 22: 354-352, Nat.Biomed. Res. Foundation, Washington, D.C. (1978).

A “hedgehog protein” or “hedgehog polypeptide”, as the terms are usedinterchangeably, of the invention is defined in terms of having at leasta portion that consists of the consensus amino acid sequence of SEQ IDNO: 4. The term also means a hedgehog polypeptide, or a functionalvariant of a hedgehog polypeptide, or homolog of a hedgehog polypeptide,or functional variant, which has biological activity. In particular, theterms encompasses preparations of hedgehog proteins and peptidylfragments thereof, both agonist and antagonist forms as the specificcontext will make clear. As used herein the term “bioactive fragment ofa hedgehog protein” refers to a fragment of a full-length hedgehogpolypeptide, wherein the fragment specifically agonizes or antagonizesinductive events mediated by wild-type hedgehog proteins. The hedgehogbiactive fragment preferably is a soluble extracellular portion of ahedgehog protein, where solubility is with reference to physiologicallycompatible solutions. Exemplary bioactive fragments are described in PCTpublications WO 95/18856 and WO 96/17924. In preferred embodiments, thehedgehog polypeptides of the present invention bind to the patchedprotein.

The term “corresponds to”, when referring to a particular polypeptide ornucleic acid sequence is meant to indicate that the sequence of interestis identical or homologous to the reference sequence to which it is saidto correspond.

The terms “peptide(s)”, “protein(s)” and “polypeptide(s)” are usedinterchangeably herein. The terms “polynucleotide sequence” and“nucleotide sequence” are also used interchangeably herein. The terms“Hedgehog fragment” and “Hedgehog N-terminal fragment” are usedinterchangeably with “Hedgehog”.

A hedgehog molecule has “biological activity” if it has at least one ofthe following properties: (i) the molecule meets the hedgehog consensuscriteria as defined herein (SEQ ID NO: 4) and has the ability to bind toits receptor, patched or it encodes, upon expression, a polypeptide thathas this characteristic; (ii) the molecule meets the hedgehog consensuscriteria as defined herein or it encodes, upon expression, a polypeptidethat has this characteristic; and (iii) it may induce alkalinephosphatase activity in C3H10T1/2 cells. Generally, any protein has“biological activity” if the protein has in vitro effects, properties,or characteristics that persons having ordinary skill in the art wouldrecognize as being representative of, commensurate with, or reasonablypredictive of, the protein's in vivo effects.

The term “hydrophobic” refers to the tendency of chemical moieties withnonpolar atoms to interact with each other rather than water or otherpolar atoms. Materials that are “hydrophobic” are, for the most part,insoluble in water. Natural products with hydrophobic properties includelipids, fatty acids, phospholipids, sphingolipids, acylglycerols, waxes,sterols, steroids, terpenes, prostaglandins, thromboxanes, leukotrienes,isoprenoids, retenoids, biotin, and hydrophobic amino acids such astryptophan, phenylalanine, isoleucine, leucine, valine, methionine,alanine, proline, and tyrosine. A chemical moiety is also hydrophobic orhas hydrophobic properties if its physical properties are determined bythe presence of nonpolar atoms. The term includes lipophilic groups.

The term “lipophilic group”, in the context of being attached to apolypeptide, refers to a group having high hydrocarbon content therebygiving the group high affinity to lipid phases. A lipophilic group canbe, for example, a relatively long chain alkyl or cycloalkyl (preferablyn-alkyl) group having approximately 7 to 30 carbons. The alkyl group mayterminate with a hydroxy or primary amine “tail”. To further illustrate,lipophilic molecules include naturally-occurring and synthetic aromaticand non-aromatic moieties such as fatty acids, esters and alcohols,other lipid molecules, cage structures such as adamantane andbuckminsterfullerenes, and aromatic hydrocarbons such as benzene,perylene, phenanthrene, anthracene, naphthalene, pyrene, chrysene, andnaphthacene.

The phrase “internal amino acid” means any amino acid in a peptidesequence that is neither the N-terminal amino acid nor the C-terminalamino acid.

The phrase “surface amino acid” means any amino acid that is exposed tosolvent when a protein is folded in its native form.

The phrase “extracellular signaling protein” means any protein that iseither secreted from a cell, or is tethered to the outside of a cell,and upon binding to the receptor for that protein on a target celltriggers a response in the target cell.

An “effective amount” of, e.g., a hedgehog polypeptide, with respect tothe subject methods of treatment, refers to an amount of polypeptide ina preparation which, when applied as part of a desired dosage regimenbrings about, e.g., a change in the rate of cell proliferation and/orthe state of differentiation of a cell and/or rate of survival of a cellaccording to clinically acceptable standards for the disorder to betreated or the cosmetic purpose.

A “patient” or “subject” to be treated by the subject method can meaneither a human or non-human animal.

The “growth state” of a cell refers to the rate of proliferation of thecell and the state of differentiation of the cell.

Practice of the present invention will employ, unless indicatedotherwise, conventional techniques of cell biology, cell culture,molecular biology, microbiology, recombinant DNA, protein chemistry, andimmunology, which are within the skill of the art. Such techniques aredescribed in the literature.

II. General Properties of Isolated Hedgehog Proteins

The polypeptide portion of the hedgehog compositions of the subjectmethod can be generated by any of a variety of techniques, includingpurification of naturally occurring proteins, recombinantly producedproteins and synthetic chemistry. Polypeptide forms of the hedgehogtherapeutics are preferably derived from vertebrate hedgehog proteins,e.g., have sequences corresponding to naturally occurring hedgehogproteins, or fragments thereof, from vertebrate organisms. However, itwill be appreciated that the hedgehog polypeptide can correspond to ahedgehog protein (or fragment thereof) which occurs in any metazoanorganism.

Isolated hedgehog proteins used in the methods of this invention arenaturally occurring or recombinant proteins of the hedgehog family andmay be obtainable from either invertebrate or from vertebrate sources(see references below). Members of the vertebrate hedgehog proteinfamily share homology with proteins encoded by the Drosophila hedgehog(hh) gene (33). To date, the combined screening of mouse genomic andcDNA libraries has identified three mammalian hh counterparts referredto as Sonic hedgehog (Shh), Indian hedgehog (Ihh), and Desert hedgehog(Dhh), which also exist in other mammals, including humans, as well asin fish and in birds. Other members include Moonrat hedgehog (Mhh), aswell as chicken Sonic hh and zebrafish Sonic hh.

Mouse and chicken Shh and mouse Ihh genes encode glycoproteins whichundergo cleavage, yielding an amino terminal fragment of about 20 kDa(See FIG. 8) and a carboxy terminal fragment of about 25 kDa. The mostpreferred 20 kDa fragment has the consensus sequence SEQ ID NO: 4 andincludes the amino acid sequences of SEQ ID NOS: 1-3. Various otherfragments that encompass the 20 kDa moiety are considered within thepresently claimed invention. Publications disclosing these sequences, aswell as their chemical and physical properties, include (34-38); PCTPatent Applications WO 95/23223 (Jessell, Dodd, Roelink and Edlund), WO95/18856 (Ingham, McMahon and Tabin) and WO 96/17924 (Beachy et al.).

Family members useful in the methods of the invention include any of thenaturally-occurring native hedgehog proteins including allelic,phylogenetic counterparts or other variants thereof, whethernaturally-sourced or produced chemically including muteins or mutantproteins, as well as recombinant forms and new, active members of thehedgehog family. Particularly useful hedgehog polypeptides include SEQID NOS: 1-4.

Isolated hedgehog polypeptides used in the method of the invention havebiological activity. The polypeptides include an amino acid sequence atleast 60%, 80%, 90%, 95%, 98%, or 99% homologous to an amino acidsequence from SEQ ID NOS; 1-4. The polypeptide can also include an aminoacid sequence essentially the same as an amino acid sequence in SEQ IDNOS: 1-4. The polypeptide is at least 5, 10, 20, 50, 100, or 150 aminoacids in length and includes at least 5, preferably at least 10, morepreferably at least 20, most preferably at least 50, 100, or 150contiguous amino acids from SEQ ID NOS: 1-4.

The preferred polypeptides of the invention include a hedgehogpolypeptide sequence as well as other N-terminal and/or C-terminal aminoacid sequence or it may include all or a fragment of a hedgehog aminoacid sequence. The isolated hedgehog polypeptide can also be arecombinant fusion protein having a first hedgehog portion and a secondpolypeptide portion, e.g., a second polypeptide portion having an aminoacid sequence unrelated to hedgehog. The second polypeptide portion canbe, e.g., histidine tag, maltose binding protein,glutathione-S-transferase, a DNA binding domain, or a polymeraseactivating domain.

Polypeptides of the invention include those which arise as a result ofthe existence of multiple genes, alternative transcription events,alternative RNA splicing events, and alternative translational andposttranslational events. The polypeptide can be made entirely bysynthetic means or can be expressed in systems, e.g., cultured cells,which result in substantially the same posttranslational modificationspresent when the protein is expressed in a native cell, or in systemswhich result in the omission of posttranslational modifications presentwhen expressed in a native cell.

In a preferred embodiment, isolated hedgehog is a hedgehog polypeptidewith one or more of the following characteristics:

-   -   (i) it has at least 30, 40, 42, 50, 60, 70, 80, 90 or 95%        sequence identity with amino acids of SEQ ID NOS: 1-4;    -   (ii) it has a cysteine or a functional equivalent as the        N-terminal end;    -   (iii) it may induce alkaline phosphatase activity in C3H10T1/2        cells;    -   (iv) it has an overall sequence identity of at least 50%,        preferably at least 60%, more preferably at least 70, 80, 90, or        95%, with a polypeptide of SEQ ID NO; 1-4;    -   (v) it can be isolated from natural sources such as mammalian        cells;    -   (vi) it can bind or interact with patched; and    -   (vii) it is hydrophobically-modified (i.e., it has at least one        hydrophobic moiety attached to the polypeptide).        III. Other Proteins

Since techniques exist for engineering a cysteine residue (or itsfunctional equivalent) into a polypeptide's primary sequence, virtuallyany protein can be converted into a hydrophobically-modified form usingthe methods described herein.

Viral receptors, cell receptors, and cell ligands are useful becausethey bind typically to cells or tissues exhibiting many copies of thereceptor. Useful viral-cell protein receptors that can be complexedtogether using the methods of this invention include ICAM1, a rhinovirusreceptor; CD2, the Epstein-Barr virus receptor; and CD4, the receptorfor human immunodeficiency virus (HIV). Other proteins include membersof the cell adhesion molecule family, such as ELAM-1 and VCAM-1 andVCAM-1b and their lymphocyte counterparts (ligands); the lymphocyteassociated antigens LFA1, LFA2 (CD2) and LFA3 (CD58), CD59 (a secondligand of CD2), members of the CD11/CD18 family and very late antigenssuch as VLA4 and their ligands.

Immunogens from a variety of pathogens (e.g., from bacterial, fungal,viral, and other eukaryotic parasites) may also be used as polypeptidesin the methods of the invention. Bacterial immunogens include, but arenot limited to, bacterial sources responsible for bacterial pneumoniaand pneumocystis pneumonia. Parasitic sources include the Plasmodiummalaria parasite. Viral sources include poxvirus (e.g, cowpox, herpessimplex, cytomegalovirus); adenoviruses; papovaviruses (e.g.,papillomavirus); parvoviruses (e.g., adeno-associated virus);retroviruses (e.g., HTLV I, HTLV II, HIV I and HIV II) and others.Immunoglobulins, or fragments thereof, may also be polypeptides that canbe modified according to the invention. One can generate monoclonal Fabfragments recognizing specific antigens using conventional methods (49)and use the individual Fab domains as functional moieties in multimericconstructs according to this invention. Other useful proteins include,gelsolin (50); cytokines, including the various interferons(interferon-α, interferon-β, and interferon-γ); the various interleukins(e.g., IL-1, -2, -3, -4, and the like); the tumor necrosis factors-α and-β; monocyte colony stimulating factor (M-CSF), granulocyte colonystimulating factor (G-CSF), granulocyte macrophage colony stimulatingfactor (GM-CSF), erythropoietin, platelet-derived growth factor (PDGF),and human and animal hormones, including growth hormone and insulin.

Generally, the structure of the modified proteins of this invention hasthe general formula: A-Cys-[Sp]-B-[Sp]-X, where A is a hydrophobicmoiety; Cys is a cysteine or a functional equivalent thereof; [Sp] is anoptional spacer peptide sequence; B is a protein (which optionally mayhave another spacer peptide sequence as shown); and X is a hydrophobicmoiety linked (optionally by way of the spacer peptide) to the aC-terminal end of the protein or another surface site of the protein,wherein the derivatized protein includes at least one of A or X. If X ischolesterol, then B may, or may not be, a hedgehog protein. As discussedabove, the purpose of the spacer is to provide separation between thehydrophobic moiety and the rest of the protein so as to make it easierfor the hydrophobic moiety (e.g., a modified N-terminal cysteine) tolink with another moiety which may be a lipid or a vesicle. The spaceris also intended to make it more difficult for the modification tointerfere with protein function. A spacer may be as small as a singleamino acid in length. Generally, prolines and glycines are preferred. Aparticularly preferred spacer sequence is derived from Sonic hedgehogand consists of the amino acid sequence: G-P-G-R.

IV. Production of Recombinant Polypeptides

The isolated polypeptides described herein can be produced by anysuitable method known in the art. Such methods range from direct proteinsynthetic methods to constructing a DNA sequence encoding isolatedpolypeptide sequences and expressing those sequences in a suitabletransformed host.

In one embodiment of a recombinant method, a DNA sequence is constructedby isolating or synthesizing a DNA sequence encoding a wild type proteinof interest. Optionally, the sequence may be mutagenized bysite-specific mutagenesis to provide functional analogs thereof. See,e.g., (40) and U.S. Pat. No. 4,588,585. Another method of constructing aDNA sequence encoding a polypeptide of interest would be by chemicalsynthesis using an oligonucleotide synthesizer. Such oligonucleotidesmay be preferably designed based on the amino acid sequence of thedesired polypeptide, and preferably selecting those codons that arefavored in the host cell in which the recombinant polypeptide ofinterest will be produced.

Standard methods may be applied to synthesize an isolated polynucleotidesequence encoding a isolated polypeptide of interest. For example, acomplete amino acid sequence may be used to construct a back-translatedgene. See Maniatis et al., supra. Further, a DNA oligomer containing anucleotide sequence coding for the particular isolated polypeptide maybe synthesized. For example, several small oligonucleotides coding forportions of the desired polypeptide may be synthesized and then ligated.The individual oligonucleotides typically contain 5′ or 3′ overhangs forcomplementary assembly.

Once assembled (by synthesis, site-directed mutagenesis, or by anothermethod), the mutant DNA sequences encoding a particular isolatedpolypeptide of interest will be inserted into an expression vector andoperatively linked to an expression control sequence appropriate forexpression of the protein in a desired host. Proper assembly may beconfirmed by nucleotide sequencing, restriction mapping, and expressionof a biologically active polypeptide in a suitable host. As is wellknown in the art, in order to obtain high expression levels of atransfected gene in a host, the gene must be operatively linked totranscriptional and translational expression control sequences that arefunctional in the chosen expression host.

The choice of expression control sequence and expression vector willdepend upon the choice of host. A wide variety of expression host/vectorcombinations may be employed. Useful expression vectors for eukaryotichosts, include, for example, vectors comprising expression controlsequences from SV40, bovine papilloma virus, adenovirus andcytomegalovirus. Useful expression vectors for bacterial hosts includeknown bacterial plasmids, such as plasmids from Esherichia coli,including pCR1, pBR322, pMB9 and their derivatives, wider host rangeplasmids, such as M13 and filamentous single-stranded DNA phages.Preferred E. coli vectors include pL vectors containing the lambda phagepL promoter (U.S. Pat. No. 4,874,702), pET vectors containing the T7polymerase promoter (Studier et al., Methods in Enzymology 185: 60-89,1990 1) and the pSP72 vector (Kaelin et al., supra). Useful expressionvectors for yeast cells, for example, include the 2 T and centromereplasmids.

In addition, any of a wide variety of expression control sequences maybe used in these vectors. Such useful expression control sequencesinclude the expression control sequences associated with structuralgenes of the foregoing expression vectors. Examples of useful expressioncontrol sequences include, for example, the early and late promoters ofSV40 or adenovirus, the lac system, the trp system, the TAC or TRCsystem, the major operator and promoter regions of phage lambda, forexample pL, the control regions of fd coat protein, the promoter for3-phosphoglycerate kinase or other glycolytic enzymes, the promoters ofacid phosphatase, e.g., Pho5, the promoters of the yeast α-mating systemand other sequences known to control the expression of genes ofprokaryotic or eukaryotic cells and their viruses, and variouscombinations thereof.

Any suitable host may be used to produce in quantity the isolatedhedgehog polypeptides described herein, including bacteria, fungi(including yeasts), plants, insects, mammals, or other appropriateanimal cells or cell lines, as well as transgenic animals or plants.More particularly, these hosts may include well known eukaryotic andprokaryotic hosts, such as strains of E. coli, Pseudomonas, Bacillus,Streptomyces, fungi, yeast (e.g., Hansenula), insect cells such asSpodoptera frugiperda (SF9), and High Five™ (see Example 1), animalcells such as Chinese hamster ovary (CHO), mouse cells such as NS/Ocells, African green monkey cells COS1, COS 7, BSC 1, BSC 40, and BMT10, and human cells, as well as plant cells.

It should be understood that not all vectors and expression controlsequences will function equally well to express a given isolatedpolypeptide. Neither will all hosts function equally well with the sameexpression system. However, one of skill in the art may make a selectionamong these vectors, expression control systems and hosts without undueexperimentation. For example, to produce isolated polypeptide ofinterest in large-scale animal culture, the copy number of theexpression vector must be controlled. Amplifiable vectors are well knownin the art. See, for example, (41) and U.S. Pat. Nos. 4,470,461 and5,122,464.

Such operative linking of a DNA sequence to an expression controlsequence includes the provision of a translation start signal in thecorrect reading frame upstream of the DNA sequence. If the particularDNA sequence being expressed does not begin with a methionine, the startsignal will result in an additional amino acid (methionine) beinglocated at the N-terminus of the product. If a hydrophobic moiety is tobe linked to the N-terminal methionyl-containing protein, the proteinmay be employed directly in the compositions of the invention.Neverthless, since the preferred N-terminal end of the protein is toconsist of a cysteine (or functional equivalent) the methionine must beremoved before use. Methods are available in the art to remove suchN-terminal methionines from polypeptides expressed with them. Forexample, certain hosts and fermentation conditions permit removal ofsubstantially all of the N-terminal methionine in vivo. Other hostsrequire in vitro removal of the N-terminal methionine. Such in vitro andin vivo methods are well known in the art.

The proteins produced by a transformed host can be purified according toany suitable method. Such standard methods include chromatography (e.g.,ion exchange, affinity, and sizing column chromatography),centrifugation, differential solubility, or by any other standardtechnique for protein purification. For immunoaffinity chromatography(See Example 1), a protein such as Sonic hedgehog may be isolated bybinding it to an affinity column comprising of antibodies that wereraised against Sonic hedgehog, or a related protein and were affixed toa stationary support. Alternatively, affinity tags such ashexahistidine, maltose binding domain, influenza coat sequence, andglutathione-S-transferase can be attached to the protein to allow easypurification by passage over an appropriate affinity column. Isolatedproteins can also be characterized physically using such techniques asproteolysis, nuclear magnetic resonance, and X-ray crystallography.

A. Production of Fragments and Analogs

Fragments of an isolated protein (e.g., fragments of SEQ ID NOS: 1-4)can also be produced efficiently by recombinant methods, by proteolyticdigestion, or by chemical synthesis using methods known to those ofskill in the art. In recombinant methods, internal or terminal fragmentsof a polypeptide can be generated by removing one or more nucleotidesfrom one end (for a terminal fragment) or both ends (for an internalfragment) of a DNA sequence which encodes for the isolated hedgehogpolypeptide. Expression of the mutagenized DNA produces polypeptidefragments. Digestion with “end nibbling” endonucleases can also generateDNAs which encode an array of fragments. DNAs which encode fragments ofa protein can also be generated by random shearing, restrictiondigestion, or a combination or both. Protein fragments can be generateddirectly from intact proteins. Peptides can be cleaved specifically byproteolytic enzymes, including, but not limited to plasmin, thrombin,trypsin, chymotrypsin, or pepsin. Each of these enzymes is specific forthe type of peptide bond it attacks. Trypsin catalyzes the hydrolysis ofpeptide bonds in which the carbonyl group is from a basic amino acid,usually arginine or lysine. Pepsin and chymotrypsin catalyse thehydrolysis of peptide bonds from aromatic amino acids, such astryptophan, tyrosine, and phenylalanine. Alternative sets of cleavedprotein fragments are generated by preventing cleavage at a site whichis suceptible to a proteolytic enzyme. For instance, reaction of theε-amino acid group of lysine with ethyltrifluorothioacetate in mildlybasic solution yields blocked amino acid residues whose adjacent peptidebond is no longer susceptible to hydrolysis by trypsin. Proteins can bemodified to create peptide linkages that are susceptible to proteolyticenzymes. For instance, alkylation of cysteine residues withβ-haloethylamines yields peptide linkages that are hydrolyzed by trypsin(51). In addition, chemical reagents that cleave peptide chains atspecific residues can be used. For example, cyanogen bromide cleavespeptides at methionine residues (52). Thus, by treating proteins withvarious combinations of modifiers, proteolytic enzymes and/or chemicalreagents, the proteins may be divided into fragments of a desired lengthwith no overlap of the fragments, or divided into overlapping fragmentsof a desired length.

Fragments can also be synthesized chemically using techniques known inthe art such as the Merrifield solid phase F moc or t-Boc chemistry.Merrifield, Recent Progress in Hormone Research 23: 451 (1967)

Examples of prior art methods which allow production and testing offragments and 5 analogs are discussed below. These, or analogous methodsmay be used to make and screen fragments and analogs of an isolatedpolypeptide (e.g., hedgehog) which can be shown to have biologicalactivity. An exemplary method to test whether fragments and analogs ofhedgehog have biological activity is found in Example 3.

B. Production of Altered DNA and Peptide Sequences: Random Methods

Amino acid sequence variants of a protein (such as variants of SEQ IDNOS: 1-4) can be prepared by random mutagenesis of DNA which encodes theprotein or a particular portion thereof. Useful methods include PCRmutagenesis and saturation mutagenesis. A library of random amino acidsequence variants can also be generated by the synthesis of a set ofdegenerate oligonucleotide sequences. Methods of generating amino acidsequence variants of a given protein using altered DNA and peptides arewell-known in the art. The following examples of such methods are notintended to limit the scope of the present invention, but merely serveto illustrate representative techniques. Persons having ordinary skillin the art will recognize that other methods are also useful in thisregard.

PCR Mutagenesis: Briefly, Taq polymerase (or another polymerase) is usedto introduce random mutations into a cloned fragment of DNA (42). PCRconditions are chosen so that the fidelity of DNA synthesis is reducedby Taq DNA polymers using, for instance, a dGTP/dATP ratio of five andadding Mn²⁺ to the PCR reaction. The pool of amplified DNA fragments isinserted into appropriate cloning vectors to provide random mutantlibraries.

Saturation Mutagenesis: One method is described generally in (43).Briefly, the technique includes generation of mutations by chemicaltreatment or irradiation of single stranded DNA in vitro, and synthesisof a cDNA strand. The mutation frequency is modulated by the severity ofthe treatment and essentially all possible base substitutions can beobtained.

Degenerate Oligonucleotide Mutagenesis: A library of homologous peptidescan be generated from a set of degenerate oligonucleotide sequences.Chemical synthesis of degenerate sequences can by performed in anautomatic DNA synthesizer, and the synthetic genes are then ligated intoan appropriate expression vector. See for example (44, 45) and Itakuraet al., Recombinant DNA, Proc. 3rd Cleveland Symposium onMacromolecules, pp. 273-289 (A. G. Walton, ed.), Elsevier, Amsterdam,1981.

C. Production of Altered DNA and Peptide Sequences: Directed Methods

Non-random, or directed, mutagenesis provides specific sequences ormutations in specific portions of a polynucleotide sequence that encodesan isolated polypeptide, to provide variants which include deletions,insertions, or substitutions of residues of the known amino acidsequence of the isolated polypeptide. The mutation sites may be modifiedindividually or in series, for instance by: (1) substituting first withconserved amino acids and then with more radical choices depending onthe results achieved; (2) deleting the target residue; or (3) insertingresidues of the same or a different class adjacent to the located site,or combinations of options 1-3.

Clearly, such site-directed methods are one way in which an N-terminalcysteine (or a functional equivalent) can be introduced into a givenpolypeptide sequence to provide the attachment site for a hydrophobicmoiety.

Alanine scanning Mutagenesis: This method locates those residues orregions of a desired protein that are preferred locations formutagenesis (46). In alanine screening, a residue or group of targetresidues are selected and replaced by alanine. This replacement canaffect the interaction of the amino acid with neighboring amino acidsand/or with the surrounding aqueous or membrane environment. Thosehaving functional sensitivity to the substitutions are then refined byintroducing further or other variants at, or for, the sites ofsubstitution.

Oligonucleotide-Mediated Mutagenesis: One version of this method may beused to prepare substitution, deletion, and insertion variants of DNA(47). Briefly, the desired DNA is altered by hybridizing anoligonucleotide primer encoding a DNA mutation to a DNA template whichtypically is the single stranded form of a plasmid or phage containingthe unaltered or wild type DNA sequence template of the desired protein(e.g., the Hedgehog protein). After hybridization, a DNA polymerase isused to make the second and complementary strand of DNA of the templatethat will incorporate the oligonucleotide primer, and will code for theselected alteration in the desired DNA sequence. Generally,oligonucleotides of at least 25 nucleotides in length are used. Anoptimal oligonucleotide will have 12 to 15 nucleotides that arecompletely complementary to the template on either side of the mutation.This ensures that the oligonucleotide will hybridize properly to thesingle-stranded DNA template molecule.

Cassette Mutagenesis: This method (48) requires a plasmid or othervector that contains the protein subunit DNA to be mutated. The codon(s)in the protein subunit DNA are identified and there is inserted a uniquerestriction endonuclease site on each side of the identified mutationsite(s), using the above-described oligonucleotide-directed mutagenesismethod. The plasmid is then cut at these sites to linearize it. Adouble-stranded oligonucleotide encoding the sequence of the DNA betweenthe restriction sites but containing the desired mutation(s) issynthesized using standard procedures. The two strands are synthesizedseparately and then hybridized together using standard methods. Thisdouble-stranded oligonucleotide is the “cassette” and it has 3′ and 5′ends that are compatible with the ends of the linearized plasmid so thatit can be directly ligated therein. The plasmid now contains the mutateddesired protein subunit DNA sequence.

Combinatorial Mutagenesis: In one version of this method (Ladner et al.,WO 88/06630), the amino acid sequences for a group of homologs or otherrelated proteins are aligned, preferably to promote the highest homologypossible. All of the amino acids which appear at a given position of thealigned sequences can be selected to create a degenerate set ofcombinatorial sequences. The variegated library is generated bycombinatorial mutagenesis at the nucleic acid level, and is encoded by avariegated gene library. For instance, a mixture of syntheticoligonucleotides can be ligated enzymically into the gene sequence suchthat the degenerate set of potential sequences are expressible asindividual peptides, or alternatively, as a set of proteins containingthe entire set of degenerate sequences.

D. Other Variants of Isolated Polypeptides

Included in the invention are isolated molecules that are: allelicvariants, natural mutants, induced mutants, and proteins encoded by DNAthat hybridizes under high or low stringency conditions to a nucleicacid which encodes a polypeptide such as the N-terminal fragment ofSonic hedgehog (SEQ ID NO: 1) and polypeptides bound specifically byantisera to hedgehog peptides, especially by antisera to an active siteor binding site of hedgehog. All variants described herein are expectedto: (i) retain the biological function of the original protein and (ii)retain the ability to link to a hydrophobic moiety (e.g, a lipid).

The methods of the invention also feature uses of fragments, preferablybiologically active fragments, or analogs of an isolated peptide such ashedgehog. Specifically, a biologically active fragment or analog is onehaving any in vivo or in vitro activity which is characteristic of thepeptide shown in SEQ ID NOS: 1-4 or of other naturally occurringisolated hedgehog. Most preferably, the hydrophobically-modifiedfragment or analog has at least 10%, preferably 40% or greater, or mostpreferably at least 90% of the activity of Sonic hedgehog (See Example3) in any in vivo or in vitro assay.

Analogs can differ from naturally occurring isolated protein in aminoacid sequence or in ways that do not involve sequence, or both. The mostpreferred polypeptides of the invention have preferred non-sequencemodifications that include in vivo or in vitro chemical derivatization(e.g., of their N-terminal end), as well as possible changes inacetylation, methylation, phosphorylation, amidation, carboxylation, orglycosylation.

Other analogs include a protein such as Sonic hedgehog or itsbiologically active fragments whose sequences differ from the wild typeconsensus sequence (e.g., SEQ ID NO: 4) by one or more conservativeamino acid substitutions or by one or more non conservative amino acidsubstitutions, or by deletions or insertions which do not abolish theisolated protein's biological activity. Conservative substitutionstypically include the substitution of one amino acid for another withsimilar characteristics such as substitutions within the followinggroups: valine, alanine and glycine; leucine and isoleucine; asparticacid and glutamic acid; asparagine and glutamine; serine and threonine;lysine and arginine; and phenylalanine and tyrosine. The non-polarhydrophobic amino acids include alanine, leucine, isoleucine, valine,proline, phenylalanine, tryptophan, and methionine. The polar neutralamino acids include glycine, serine, threonine, cysteine, tyrosine,asparagine, and glutamine. The positively charged (basic) amino acidsinclude arginine, lysine, and histidine. The negatively charged (acidic)amino acids include aspartic acid and glutamic acid. Other conservativesubstitutions can be readily known by workers of ordinary skill. Forexample, for the amino acid alanine, a conservative substitution can betaken from any one of D-alanine, glycine, beta-alanine, L-cysteine, andD-cysteine. For lysine, a replacement can be any one of D-lysine,arginine, D-arginine, homo-arginine, methionine, D-methionine,ornithine, or D-ornithine.

Generally, substitutions that may be expected to induce changes in thefunctional properties of isolated polypeptides are those in which: (i) apolar residue, e.g., serine or threonine, is substituted for (or by) ahydrophobic residue, e.g., leucine, isoleucine, phenylalanine, oralanine; (ii) a cysteine residue is substituted for (or by) any otherresidue (See Example 10); (iii) a residue having an electropositive sidechain, e.g., lysine, arginine or histidine, is substituted for (or by) aresidue having an electronegative side chain, e.g., glutamic acid oraspartic acid; or (iv) a residue having a bulky side chain, e.g.,phenylalanine, is substituted for (or by) one not having such a sidechain, e.g., glycine.

Other analogs used within the methods of the invention are those withmodifications which increase peptide stability. Such analogs maycontain, for example, one or more non-peptide bonds (which replace thepeptide bonds) in the peptide sequence. Also included are: analogs thatinclude residues other than naturally occurring L-amino acids, such asD-amino acids or non-naturally occurring or synthetic amino acids suchas beta or gamma amino acids and cyclic analogs. Incorporation of D-instead of L-amino acids into the isolated hedgehog polypeptide mayincrease its resistance to proteases. See, U.S. Pat. No. 5,219,990supra.

The term “fragment”, as applied to an isolated hedgehog analog, can beas small as a single amino acid provided that it retains biologicalactivity. It may be at least about 20 residues, more typically at leastabout 40 residues, preferably at least about 60 residues in length.Fragments can be generated by methods known to those skilled in the art.The ability of a candidate fragment to exhibit isolated hedgehogbiological activity can be also assessed by methods known to thoseskilled in the art as described herein.

V. Making Hydrophobic Derivatives

The inventors have discovered that increasing the overall hydrophobicnature of a signaling protein, such as a hedgehog protein, increases thebiological activity of the protein. The potency of a signaling proteinsuch as hedgehog can be increased by: (a) chemically modifying, such asby adding a hydrophobic moiety to, the sulfhydryl and/or to the α-amineof the N-terminal cysteine (Examples 8 and 9); (b) replacing theN-terminal cysteine with a hydrophobic amino acid (Example 10); or (c)replacing the N-terminal cysteine with a different amino acid and thenchemically modifying the substituted residue so as to add a hydrophobicmoiety at the site of the substitution.

Additionally, modification of a protein such as hedgehog protein at aninternal residue on the surface of the protein with a hydrophobic moietyby: (a) replacing the internal residue with a hydrophobic amino acid; or(b) replacing the internal residue with a different amino acid and thenchemically modifying the substituted residue so as to add a hydrophobicmoiety at the site of the substitution (See Example 10), will retain orenhance the biological activity of the protein.

Additionally, modification of a protein such as a hedgehog protein atthe C-terminus with a hydrophobic moiety by: (a) replacing theC-terminal residue with a hydrophobic amino acid; or (b) replacing theC-terminal residue with a different amino acid and then chemicallymodifying the substituted residue so as to add a hydrophobic moiety atthe site of the substitution, will retain or enhance the biologicalactivity of the protein.

There are a wide range of lipophilic moieties with which hedgehogpolypeptides can be derivatived. A lipophilic group can be, for example,a relatively long chain alkyl or cycloalkyl (preferably n-alkyl) grouphaving approximately 7 to 30 carbons. The alkyl group may terminate witha hydroxy or primary amine “tail”. To further illustrate, lipophilicmolecules include. naturally-occurring and synthetic aromatic andnon-aromatic moieties such as fatty acids, esters and alcohols, otherlipid molecules, cage structures such as adamantane andbuckminsterfullerenes, and aromatic hydrocarbons such as benzene,perylene, phenanthrene, anthracene, naphthalene, pyrene, chrysene, andnaphthacene.

Particularly useful as lipophilic molecules are alicyclic hydrocarbons,saturated and unsaturated fatty acids and other lipid and phospholipidmoieties, waxes, cholesterol, isoprenoids, terpenes and polyalicyclichydrocarbons including adamantane and buckminsterfullerenes, vitamins,polyethylene glycol or oligoethylene glycol, (C1-C18)-alkyl phosphatediesters, —O—CH2—CH(OH)—O—(C12-C18)-alkyl, and in particular conjugateswith pyrene derivatives. The lipophilic moiety can be a lipophilic dyesuitable for use in the invention include, but are not limited to,diphenylhexatriene, Nile Red, N-phenyl-1-naphthylamine, Prodan,Laurodan, Pyrene, Perylene, rhodamine, rhodamine B,tetramethylrhodamine, Texas Red, sulforhodamine,1,1′-didodecyl-3,3,3′,3′tetramethylindocarbocyanine perchlorate,octadecyl rhodamine B and the BODIPY dyes available from MolecularProbes Inc.

Other exemplary lipophilic moietites include aliphatic carbonyl radicalgroups include 1- or 2-adamantylacetyl, 3-methyladamant-1-ylacetyl,3-methyl-3-bromo-1-adamantylacetyl, 1-decalinacetyl, camphoracetyl,camphaneacetyl, noradamantylacetyl, norbornaneacetyl,bicyclo[2.2.2.]-oct-5-eneacetyl,1-methoxybicyclo[2.2.2.]-oct-5-ene-2-carbonyl,cis-5-norbornene-endo-2,3-dicarbonyl, 5-norbornen-2-ylacetyl,(1R)-(-)-myrtentaneacetyl, 2-norbornaneacetyl,anti-3-oxo-tricyclo[2.2.1.0<2,6>]-heptane-7-carbonyl, decanoyl,dodecanoyl, dodecenoyl, tetradecadienoyl, decynoyl or dodecynoyl.

Structures of exemplary hydrophobic modifications are shown in FIG. 12.If an appropriate amino acid is not available at a specific position,site-directed mutagenesis can be used to place a reactive amino acid atthat site. Reactive amino acids include cysteine, lysine, histidine,aspartic acid, glutamic acid, serine, threonine, tyrosine, arginine,methionine, and tryptophan. Mutagenesis could be used to place thereactive amino acid at the N- or C-terminus or at an internal position.

For example, we have discovered that it is possible to chemically modifyan N-terminal cysteine of a biologically active protein, such as ahedgehog protein, or eliminate the N-terminal cysteine altogether andstill retain the protein's biological activity, provided that themodified or substituted chemical moiety is hydrophobic. The inventorshave found that enhancement of hedgehog's biological activity roughlycorrelates with the hydrophobicity of the modification. In addition toenhancing the protein's activity, modifying or replacing the N-terminalcysteine eliminates unwanted cross reactions and/or modifications of thecysteine that can occur during production, purification, formulation,and storage of the protein. The thiol of an N-terminal cysteine is veryreactive due to its proximity to the α-amine which lowers the pKa of thecysteine and increases proton dissociation and formation of the reactivethiolate ion at neutral or acid pH.

We have demonstrated that replacement of the N-terminal cysteine ofhedgehog with a hydrophobic amino acid results in a protein withincreased potency in a cell-based signaling assay. By replacing thecysteine, this approach eliminates the problem of suppressing otherunwanted modifications of the cysteine that can occur during theproduction, purification, formulation, and storage of the protein. Thegenerality of this approach is supported by our finding that threedifferent hydrophobic amino acids, phenylalanine, isoleucine, andmethionine, each give a more active form of hedgehog. Therefore,replacement of the cysteine with any other hydrophobic amino acid shouldresult in an active protein. Furthermore, since we have found acorrelation between the hydrophobicity of an amino acid or chemicalmodification and the potency of the corresponding modified protein inthe C3H10T1/2 assay (e.g. Phe>Met, long chain length fatty acids>shortchain length), it could be envisioned that adding more than onehydrophobic amino acid to the hedgehog sequence would increase thepotency of the protein beyond that achieved with a single amino acidaddition. Indeed, addition of two consecutive isoleucine residues to theN-terminus of human Sonic hedgehog results in an increase in potency inthe C3H10T1/2 assay as compared to the mutant with only a singleisoleucine added (See Example 10). Thus, adding hydrophobic amino acidsat the N- or C-terminus of a hedgehog protein, in a surface loop, orsome combination of positions would be expected to give a more activeform of the protein. The substituted amino acid need not be one of the20 common amino acids. Methods have been reported for substitutingunnatural amino acids at specific sites in proteins (78, 79) and thiswould be advantageous if the amino acid was more hydrophobic incharacter, resistant to proteolytic attack, or could be used to furtherdirect the hedgehog protein to a particular site in vivo that would makeits activity more potent or specific. Unnatural amino acids can beincorporated at specific sites in proteins during in vitro translation,and progress is being reported in creating in vivo systems that willallow larger scale production of such modified proteins.

It is unexpected that a protein, such as an hedgehog protein, modifiedaccording to the invention, would retain its biological activity. First,the N-terminal cysteine is conserved in all known hedgehog proteinsequences including fish, frog, insect, bird, and mammals. Therefore, itis reasonable to expect that the free sulfhydryl of the N-terminalcysteine is important to the protein's structure or activity. Second,hedgehog proteins lacking an N-terminal cysteine, due to proteolyticcleavage or mutation to hydrophilic amino acids (e.g., aspartic acid orhistidine) are inactive in a the cell-based C3H10T1/2 assay, such asthat described in Example 3.

There are many modifications of the N-terminal cysteine which protectthe thiol and append a hydrophobic moiety. These modifications arediscussed in more detail below. One of skill in the art is capable ofdetermining which modification is most appropriate for a particulartherapeutic use. Factors affecting such a determination include cost andease of production, purification and formulation, solubility, stability,potency, pharmacodynamics and kinetics, safety, immunogenicity, andtissue targeting.

A. Chemical Modifications of Primary Amino Acid Sequence

The chemical modification of the N-terminal cysteine to protect thethiol, with concomitant activation by a hydrophobic moiety, can becarried out in numerous ways by someone skilled in the art. Thesulfhydryl moiety, with the thiolate ion as the active species, is themost reactive functional group in a protein. There are many reagentsthat react faster with the thiol than any other groups. See Chemistry ofProtein Conjugation and Cross-Linking (S. S. Wong, CRC Press, BocaRaton, Fla., 1991). The thiol of an N-terminal cysteine, such as foundin all hedgehog proteins, would be expected to be more reactive thaninternal cysteines within the sequence. This is because the closeproximity to the α-amine will lower the pKa of the thiol resulting in agreater degree of proton dissociation to the reactive thiolate ion atneutral or acid pH. In addition, the cysteine at the N-terminus of thestructure is more likely to be exposed than the other two cysteines inthe hedgehog sequence that are found buried in the protein structure. Wehave shown that the N-terminal cysteine is the only amino acid modifiedafter a 1 h reaction with N-ethylmaleimide at pH 5.5 (See Example 9),and after a 18 h reaction with N-isopropyliodoacetamide at pH 7.0 (SeeExample 9). Other examples of such methods would be reaction with otherα-haloacetyl compounds, organomercurials, disulfide reagents, and otherN-substituted maleimides. Numerous hydrophobic derivatives of theseactive species are available commercially (e.g., ethyl iodoacetate(Aldrich, Milwaukee Wis.), phenyl disulfide (Aldrich), andN-pyrenemaleimide (Molecular Probes, Eugene Oreg.)) or could besynthesized readily (e.g., N-alkyliodoacetamides (84), N-alkylmaleimides(85), and organomercurials (86). We have shown that the N-terminalcysteine of human Sonic hedgehog can be specifically modified withN-isopropyliodoacetamide and that the hydrophobically-modified proteinis 2-fold more potent in the C3H10T1/2 assay than the unmodified protein(See Example 9). It is expected that modification of Shh with along-chain alkyl iodoacetamide derivative will result in a modifiedprotein with even greater enhancement of potency. SuchN-alkyliodoacetamides can be synthesized readily by ones skilled in theart, using commercially available starting materials.

Another aspect to the reactivity of an N-terminal cysteine is that itcan take part in reaction chemistries unique to its 1,2-aminothiolconfiguration. One example is the reaction with thioester groups to forman N-terminal amide group via a rapid S to N shift of the thioester.This reaction chemistry can couple together synthetic peptides and canbe used to add single or multiple, natural or unnatural, amino acids orother hydrophobic groups via the appropriately activated peptide.Another example, demonstrated herein, is the reaction with aldehydes toform the thiazolidine adduct. Numerous hydrophobic derivatives of thiolesters (e.g., C2-C24 saturated and unsaturated fatty acyl Coenzyme Aesters (Sigma Chemical Co., St. Louis Mo.)), aldehydes (e.g.,butyraldehyde, n-decyl aldehyde, and n-myristyl aldehyde (Aldrich)), andketones (e.g., 2-, 3-, and 4-decanone (Aldrich)) are availablecommercially or could be synthesized readily (87, 88). In a similarmanner, thiomorpholine derivatives exemplified by the 1-bromo-2-butanonechemistry described in Example 9 could be prepared from a variety ofα-haloketone starting materials (88). Because of the ease of findingalternative routes to modifying the thiol of the N-terminal cysteine, orany cysteine in a protein, we do not wish to be bound by the specificexamples demonstrated here.

The α-amine of a protein can be modified preferentially relative toother amines in a protein because its lower pKa results in higheramounts of the reactive unprotonated form at neutral or acidic pH. Wehave shown that modification of the N-terminal amine with a long chainfatty amide group, while maintaining a free cysteine thiol group,activates the hedgehog protein by as much as two orders of magnitude(See Example 8). Therefore chemistries that can be directed to reactpreferentially with the N-terminal amine would be expected to be of usein increasing the potency of the hedgehog proteins. Aryl halides,aldehydes and ketones, acid anhydrides, isocyanates, isothiocyanates,imidoesters, acid halides, N-hydroxysuccinimidyl (e.g.,sulfo-NHS-acetate), nitrophenyl esters, acylimidazoles, and otheractivated esters are among those known to react with amine functions.

By replacing the N-terminal cysteine of hedgehog with certain otheramino acids, other chemical methods can be used to add a hydrophobicmoiety to the N-terminus. One example is to place a serine or threonineat the N-terminus, oxidize this amino acid to form an aldehyde, and thenconjugate the protein with a chemical moiety containing a 1,2 aminothiolstructure (e.g., a cysteine). A second example would be to place ahistidine at the N-terminus to couple to a C-terminal thiocarboxylicacid.

Chemical Modification of Other Amino Acids.

There are specific chemical methods for the modification of many otheramino acids. Therefore another route for synthesizing a more active-formof hedgehog would be to chemically attach a hydrophobic moiety to anamino acid in hedgehog other than to the N-terminal cysteine. If anappropriate amino acid is not available at the desired position,site-directed mutagenesis could be used to place the reactive amino acidat that site in the hedgehog structure, whether at the N- or C-terminusor at another position. Reactive amino acids would include cysteine,lysine, histidine, aspartic acid, glutamic acid, serine, threonine,tyrosine, arginine, methionine, and tryptophan. Thus the goal ofcreating a more hydrophobic form of hedgehog could be attained by manychemical means and we do not wish to be restricted by a particularchemistry or site of modification since our results support thegenerality of this approach.

The hedgehog polypeptide can be linked to the hydrophobic moiety in anumber of ways including by chemical coupling means, or by geneticengineering.

To illustrate, there are a large number of chemical cross-linking agentsthat are known to those skilled in the art. For the present invention,the preferred cross-linking agents are heterobifunctional cross-linkers,which can be used to link the hedgehog polypeptide and hydrophobicmoiety in a stepwise manner. Heterobifunctional cross-linkers providethe ability to design more specific coupling methods for conjugating toproteins, thereby reducing the occurrences of unwanted side reactionssuch as homo-protein polymers. A wide variety of heterobifunctionalcross-linkers are known in the art. These include: succinimidyl4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC),m-Maleimidobenzoyl-N-hydroxysuccinimide ester (MBS);N-succinimidyl(4-iodoacetyl)aminobenzoate (SLAB), succinimidyl4-(p-maleimidophenyl)butyrate (SMPB),1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC);4-succinimidyloxycarbonyl-a-methyl-a-(2-pyridyldithio)-tolune (SMPT),N-succinimidyl 3-(2-pyridyldithio)propionate (SPDP), succinimidyl6-[3-(2-pyridyldithio)propionate]hexanoate (LC-SPDP). Thosecross-linking agents having N-hydroxysuccinimide moieties can beobtained as the N-hydroxysulfosuccinimide analogs, which generally havegreater water solubility. In addition, those cross-linking agents havingdisulfide bridges within the linking chain can be synthesized instead asthe alkyl derivatives so as to reduce the amount of linker cleavage invivo.

In addition to the heterobifunctional cross-linkers, there exists anumber of other cross-linking agents including homobifunctional andphotoreactive cross-linkers. Disuccinimidyl suberate (DSS),bismaleimidohexane (BMH) and dimethylpimelimidate-2 HCl (DMP) areexamples of useful homobifunctional cross-linking agents, andbis-[β-(4-azidosalicylamido)ethyl]disulfide (BASED) andN-succinimidyl-6(4′-azido-2′-nitrophenyl-amino)hexanoate (SANPAH) areexamples of useful photoreactive cross-linkers for use in thisinvention. For a recent review of protein coupling techniques, see Meanset al. (1990) Bioconjugate Chemistry 1:2-12, incorporated by referenceherein.

One particularly useful class of heterobifunctional cross-linkers,included above, contain the primary amine reactive group,N-hydroxysuccinimide (NHS), or its water soluble analogN-hydroxysulfosuccinimide (sulfo-NHS). Primary amines (lysine epsilongroups) at alkaline pH's are unprotonated and react by nucleophilicattack on NHS or sulfo-NHS esters. This reaction results in theformation of an amide bond, and release of NHS or sulfo-NHS as aby-product.

Another reactive group useful as part of a heterobifunctionalcross-linker is a thiol reactive group. Common thiol reactive groupsinclude maleimides, halogens, and pyridyl disulfides. Maleimides reactspecifically with free sulfhydryls (cysteine residues) in minutes, underslightly acidic to neutral (pH 6.5-7.5) conditions. Halogens (iodoacetylfunctions) react with —SH groups at physiological pH's. Both of thesereactive groups result in the formation of stable thioether bonds.

The third component of the heterobifunctional cross-linker is the spacerarm or bridge. The bridge is the structure that connects the tworeactive ends. The most apparent attribute of the bridge is its effecton steric hindrance. In some instances, a longer bridge can more easilyspan the distance necessary to link two complex biomolecules. Forinstance, SMPB has a span of 14.5 angstroms.

Preparing protein-protein conjugates using heterobifunctional reagentsis a two-step process involving the amine reaction and the sulfhydrylreaction. For the first step, the amine reaction, the protein chosenshould contain a primary amine. This can be lysine epsilon amines or aprimary alpha amine found at the N-terminus of most proteins. Theprotein should not contain free sulfhydryl groups. In cases where bothproteins to be conjugated contain free sulfhydryl groups, one proteincan be modified so that all sulfhydryls are blocked using for instance,N-ethylmaleimide (see Partis et al. (1983) J. Pro. Chem. 2:263,incorporated by reference herein). Ellman's Reagent can be used tocalculate the quantity of sulfhydryls in a particular protein (see forexample Ellman et al. (1958) Arch. Biochem. Biophys. 74:443 and Riddleset al. (1979) Anal. Biochem. 94:75, incorporated by reference herein).

The reaction buffer should be free of extraneous amines and sulfhydryls.The pH of the reaction buffer should be 7.0-7.5. This pH range preventsmaleimide groups from reacting with amines, preserving the maleimidegroup for the second reaction with sulfhydryls.

The NHS-ester containing cross-linkers have limited water solubility.They should be dissolved in a minimal amount of organic solvent (DMF orDMSO) before introducing the cross-linker into the reaction mixture. Thecross-linker/solvent forms an emulsion which will allow the reaction tooccur.

The sulfo-NHS ester analogs are more water soluble, and can be addeddirectly to the reaction buffer. Buffers of high ionic strength shouldbe avoided, as they have a tendency to “salt out” the sulfo-NHS esters.To avoid loss of reactivity due to hydrolysis, the cross-linker is addedto the reaction mixture immediately after dissolving the proteinsolution.

The reactions can be more efficient in concentrated protein solutions.The more alkaline the pH of the reaction mixture, the faster the rate ofreaction. The rate of hydrolysis of the NHS and sulfo-NHS esters willalso increase with increasing pH. Higher temperatures will increase thereaction rates for both hydrolysis and acylation.

Once the reaction is completed, the first protein is now activated, witha sulfhydryl reactive moiety. The activated protein may be isolated fromthe reaction mixture by simple gel filtration or dialysis. To carry outthe second step of the cross-linking, the sulfhydryl reaction, thelipophilic group chosen for reaction with maleimides, activatedhalogens, or pyridyl disulfides must contain a free sulfhydryl.Alternatively, a primary amine may be modified with to add a sulfhydryl.

In all cases, the buffer should be degassed to prevent oxidation ofsulfhydryl groups. EDTA may be added to chelate any oxidizing metalsthat may be present in the buffer. Buffers should be free of anysulfhydryl containing compounds.

Maleimides react specifically with —SH groups at slightly acidic toneutral pH ranges (6.5-7.5). A neutral pH is sufficient for reactionsinvolving halogens and pyridyl disulfides. Under these conditions,maleimides generally react with —SH groups within a matter of minutes.Longer reaction times are required for halogens and pyridyl disulfides.

The first sulfhydryl reactive-protein prepared in the amine reactionstep is mixed with the sulfhydryl-containing lipophilic group under theappropriate buffer conditions. The conjugates can be isolated from thereaction mixture by methods such as gel filtration or by dialysis.

Exemplary activated lipophilic moieties for conjugation include:N-(1-pyrene)maleimide; 2,5-dimethoxystilbene-4′-maleimide,eosin-5-maleimide; fluorescein-5-maleimide;N-(4-(6-dimethylamino-2-benzofuranyl)phenyl)maleimide;benzophenone-4-maleimide; 4-dimethylaminophenylazophenyl-4′-maleimide(DABMI), tetramethylrhodamine-5-maleimide,tetramethylrhodamine-6-maleimide, Rhodamine Red™ C2 maleimide,N-(5-aminopentyl)maleimide, trifluoroacetic acid salt,N-(2-aminoethyl)maleimide, trifluoroacetic acid salt, Oregon Green™ 488maleimide,N-(2-((2-(((4-azido-2,3,5,6-tetrafluoro)benzoyl)amino)ethyl)dithio)ethyl)maleimide(TFPAM-SS1),2-(1-(3-dimethylaminopropyl)-indol-3-yl)-3-(indol-3-yl)maleimide(bisindolylmaleimide; GF 109203X), BODIPY® FL N-(2-aminoethyl)maleimide,N-(7-dimethylamino-4-methylcoumarin-3-yl)maleimide (DACM), Alexa™ 488 C5maleimide, Alexa™ 594 C5 maleimide, sodium saltN-(1-pyrene)maleimide,2,5-dimethoxystilbene-4′-maleimide, eosin-5-maleimide,fluorescein-5-maleimide,N-(4-(6-dimethylamino-2-benzofuranyl)phenyl)maleimide,benzophenone-4-maleimide, 4-dimethylaminophenylazophenyl-4′-maleimide,1-(2-maleimidylethyl)-4-(5-(4-methoxyphenyl)oxazol-2-yl)pyridiniummethanesulfonate, tetramethylrhodamine-5-maleimide,tetramethylrhodamine-6-maleimide, Rhodamine Red™ C2 maleimide,N-(5-aminopentyl)maleimide, N-(2-aminoethyl)maleimide,N-(2-((2-(((4-azido-2,3,5,6-tetrafluoro)benzoyl)amino)ethyl)dithio)ethyl)maleimide,2-(1-(3-dimethylaminopropyl)-indol-3-yl)-3-(indol-3-yl) maleimide,N-(7-dimethylamino-4-methylcoumarin-3-yl)maleimide (DACM),11H-Benzo[a]fluorene, Benzo[a]pyrene.

In one embodiment, the hedgehog polypeptide can be derivatived usingpyrene maleimide, which can be purchased from Molecular Probes (Eugene,Oreg.), e.g., N-(1-pyrene)maleimide or 1-pyrenemethyl iodoacetate (PMIAester). As illustrated in FIG. 1, the pyrene-derived hedgehog proteinhad an activity profile indicating that it was nearly 2 orders ofmagnitude more active than the unmodified form of the protein.

B. Making Hydrophobic Peptide Derivatives

According to the invention, the protein can also be modified using ahydrophobic peptide. As used herein, the term “peptide” includes asequence of at least one amino acid residue. Preferably, the peptide hasa length between one amino acid and 18-26 amino acids, the latter beingthe typical length of a membrane spanning segment of a protein. Tocreate a peptide with hydrophobic character, the amino acids areselected predominantly from the following hydrophobic amino acids:phenylalanine, isoleucine, leucine, valine, methionine, tryptophan,alanine, proline, and tyrosine. The hydrophobic peptide can also containunnatural amino acid analogs with hydrophobic character or D-aminoacids, peptoid bonds, N-terminal acetylation or other features thatdecrease the peptide's susceptibility to proteolysis. Methods forsubstituting unnatural amino acids at specific sites in proteins areknown (78, 79).

Generally, a hydrophobic peptide is appended to various sites on aprotein. One site can be the N-terminal residue. Alternatively, thehydrophobic peptide is substituted in place of the N-terminal residue.In another embodiment, a hydrophobic peptide is appended to theC-terminus of the protein. Alternatively, the hydrophobic peptide issubstituted in place of the C-terminal residue. The C-terminus can bethe native C-terminal amino acid but it may also be the C-terminus of atruncated protein so that the hydrophobic peptide is appended to thefinal C-terminal amino acid of the truncated form, which is stillreferred to as the “C-terminus”. A truncated hedgehog protein willretain activity when up to eleven amino acids are deleted from thenative C-terminal sequence. The hydrophobic peptide may also be insertedbetween the N-terminal residue and the internal residue immediatelyadjacent to the N-terminal residue, or between the C-terminal residueand the residue immediately adjacent to the C-terminal residue, orbetween two internal residues.

In certain embodiments, the lipophilic moiety is an amphipathicpolypeptide, such as magainin, cecropin, attacin, melittin, gramicidinS, alpha-toxin of Staph. aureus, alamethicin or a synthetic amphipathicpolypeptide. Fusogenic coat proteins from viral particles can also be aconvenient source of amphipathic sequences for the subject hedgehogproteins.

C. Making Lipid Derivatives

Another form of protein encompassed by the invention is a proteinderivatized with a variety of lipid moieties. Generally, a “lipid” is amember of a heterogenous class of hydrophobic substances characterizedby a variable solubility in organic solvents and insolubility, for themost part, in water. The principal classes of lipids that areencompassed within this invention are fatty acids and sterols (e.g.,cholesterol). Derivatized proteins of the invention contain fatty acidswhich are cyclic, acyclic (i.e., straight chain), saturated orunsaturated, monocarboxylic acids. Exemplary saturated fatty acids havethe generic formula: CH₃(CH₂)n COOH. The following table lists examplesof some fatty acids that can be derivatized conveniently usingconventional chemical methods.

Table 2: Exemplary Saturated and Unsaturated Fatty Acids SaturatedAcids: CH₃(CH₂)n COOH Value of n Common Name  2 butyric acid  4 caproicacid  6 caprylic acid  8 capric acid 10 lauric acid 12 myristic acid* 14palmitic acid* 16 stearic acid* 18 arachidic acid* 20 behenic acid 22lignoceric acid Unsaturated Acids CH₃CH═CHCOOH crotonic acidCH₃(CH₂)₃CH═CH(CH₂)₇COOH myristoleic acid* CH₃(CH₂)₅CH═CH(CH₂)₇COOHpalmitoleic acid* CH₃(CH₂)₇CH═CH(CH₂)₇COOH oleic acid*CH₃(CH₂)₃(CH₂CH═CH)₂(CH₂)₇COOH linoleic acid CH₃(CH₂CH═CH)₃(CH₂)₇COOHlinolenic acid CH₃(CH₂)₃(CH₂CH═CH)₄(CH₂)₃COOH arachidonic acidThe asterisk (*) denotes the fatty acids that we found in recombinanthedgehog protein secreted from a soluble construct.

Other lipids that can be attached to the protein include branched-chainfatty acids and those of the phospholipid group such as thephosphatidylinositols (i.e., phosphatidylinositol 4-monophosphate andphosphatidylinositol 4,5-biphosphate), phosphatidycholine,phosphatidylethanolamine, phosphatidylserine, and isoprenoids such asfamesyl or geranyl groups.

We have demonstrated that lipid-modified hedgehog proteins can bepurified from either a natural source, or can be obtained by chemicallymodifying the soluble, unmodified protein. For protein purified from anatural source, we showed that when full-length human Sonic hedgehog(Shh) was expressed in insect cells and membrane-bound Shh purified fromthe detergent-treated cells using a combination of SP-Sepharosechromatography and immunoaffinity chromatography, that the purifiedprotein migrated on reducing SDS-PAGE gels as a single sharp band withan apparent mass of 20 kDa (See Example 1). The soluble andmembrane-bound Shh proteins were readily distinguishable by reversephase HPLC, where the tethered forms eluted later in the acetonitrilegradient (See Example 1 and FIG. 3). We then demonstrated that humanSonic hedgehog is tethered to cell membranes in two forms, one form thatcontains a cholesterol, and therefore is analogous to the data reportedpreviously for Drosophila hedgehog (18), and a second novel form thatcontains both a cholesterol and a palmitic acid modification. Solubleand tethered forms of Shh were analyzed by electrospray massspectrometry using a triple quadrupole mass spectrometer, equipped withan electrospray ion source (Example 1) as well as by liquidchromatography-mass spectrometry (See Example 1). The identity of theN-terminal peptide from endoproteinase Lys-C digested tethered Shh wasconfirmed by MALDI PSD mass spectrometric measurement on a MALDI time offlight mass spectrometer. The site of palmitoylation was identifiedthrough a combination of peptide mapping and sequence analysis and is atthe N-terminus of the protein (residue 1 of the sequence of the matureprotein in SEQ ID NOS: 1-4). Both tethered forms were equally as activein the C3H10T1/2 alkaline phosphatase assay, but interestingly both wereabout 30-times more potent than soluble human Shh lacking the tether(s).The lipid modifications did not significantly affect the apparentbinding affinity of Shh for its receptor, patched (FIG. 7).

We next tested the utility of the derivatized forms by assaying therelative potencies of soluble and tethered Shh alone or in the presenceof the anti-hedgehog neutralizing Mab 5E1 on C3H10T1/2 cells measuringalkaline phosphatase induction. Moreover, the relative potency ofsoluble and tethered Shh for binding to patched was assessed onpatched-transfected EBNA-293 cells by FACS analysis (Example 3).

For lipid-modified hedgehog obtained by chemically modifying thesoluble, unmodified protein, we have showed that palmitic acid and otherlipids can be added to soluble Shh to create a lipid-modified forms withincreased potency in the C3H10T1/2 assay (Example 8). We have shown(Examples 1, 2, and 8) that the thiol and α-amine on the N-terminalcysteine contribute to the lipid derivatization reaction. Withoutwishing to be bound by any particular theory, lipid modification onproteins starts with the formation of a thioester intermediate and thelipid moiety is then transferred to the α-amine of the N-terminusthrough the formation of a cyclic intermediate. Generally, therefore,the reactive lipid moiety can be in the form of thioesters of saturatedor unsaturated carboxylic acids such as a Coenzyme A thioesters. Suchmaterials and their derivatives may include, for example, commerciallyavailable Coenzyme A derivatives such as palmitoleoyl Coenzyme A,arachidoyl Coenzyme A, arachidonoyl Coenzyme A, lauroyl Coenzyme A andthe like. These materials are readily available from Sigma ChemicalCompany (St. Louis, Mo., 1998 catalog pp. 303-306).

The effect of different lipid moieties on functional activity ofhedgehog protein has been assayed (See Example 8 and FIGS. 10 and 11).Similarly, the effect of different lipid moieties on functional activityof other proteins such as those described above in Section III, may beconveniently tested using methods known to workers of ordinary skill.For instance, functional testing of gelsolin (50), various interferons(interferon-α, interferon-β and interferon-γ), the various interleukins(e.g., IL-1, -2, -3, -4, and the like), the tumor necrosis factors-α and-β, and other growth factors that are lipid-modified according to theinvention can be accomplished using well known methods.

Although we have established chemical means by which a fatty acid can beattached to the N-terminal cysteine of hedgehog proteins, it might beexpected that lipids can be attached to the same or other sites usingenzymically catalyzed reactions. Palmitoylation of proteins in vivo iscatalyzed by a class of enzymes known as palmitoyl-CoA:proteinS-palmitoyltransferases. Using purified enzymes, in vitro acylation ofprotein substrates has been demonstrated (80, 81). The substratespecificities of the palmitoyltransferase enzymes are not well defined;an analysis of palmitoylation sites of cellular and viral proteins findslittle in the way of a consensus sequence surrounding the modifiedcysteine residue, but suggests a common presence of a lysine or arginineresidue within two amino acids of the cysteine, and large, hydrophobicamino acids near the cysteine. The amino-terminal sequence of Shh,CGPGRGFG, may fit this consensus sequence and serve as a recognitionsite for palmitoylation.

As an alternative, myristoylation of the amino terminus of hedgehogproteins could be carried out using an N-myristoyl transferase (NMT), anumber of which have been well characterized in both mammals (82) and inyeast (83). A recognition site for N-myristoyltransferase could beengineered into the hedgehog N-terminal sequence to facilitaterecognition by the enzyme. Both of these strategies would require theuse of fatty acyl-coenzyme A derivatives as substrates, as are used forthe non-enzymic fatty acylation of human Sonic hedgehog described inExample 8. Alternatively, a protein with an engineered recognitionsequence may be myristoylated during expression in a suitable cell line.Another method of modifying a protein such as hedgehog with ahydrophobic moiety is to create a recognition site for the addition ofan isoprenoid group at the C-terminus of the protein. The recognitionsite for farnesyl and geranyl-geranyl addition are known and the proteinmay be modified during expression in a eukaryotic cell (Gelb et al.,Cur. Opin. Chem. Biol. 2: 40-48 (1998)).

VI. Multimeric Protein Complexes

Hydrophobically-modified proteins described herein are particularlyamenable to being made into multimeric protein complexes. Multimericprotein complexes of the invention include proteins, optionally attachedvia their hydrophobic (e.g., lipid) moiety to a vesicle. The vesicle maybe a naturally occurring biological membrane, purified away from naturalmaterial, or the vesicle may be a synthetic construction. Preferredvesicles are substantially spherical structures made of amphiphiles,e.g., surfactants or phospholipids. The lipids of these sphericalvesicles are generally organized in the form of lipids having one ormore structural layers, e.g., multilamellar vesicles (multipleonion-like shells of lipid bilayers which encompass an aqueous volumebetween the bilayers) or micelles.

In particular, liposomes are small, spherical vesicles composedprimarily of various types of lipids, phospholipids, and secondarylipophilic components. These components are normally arranged in abilayer formation, similar to the lipid arrangement of biologicalmembranes.

Typically, the polar end of a component lipid or lipid-like molecule isin contact with the surrounding solution, usually an aqueous solution,while the non-polar, hydrophobic end of the lipid or lipid-like moleculeis in contact with the non-polar, hydrophobic end of another lipid orlipid-like molecule. The resulting bilayer membrane (i.e., vesicle) isselectively permeable to molecules of a certain size, hydrophobicity,shape, and net charge. Most vesicles are lipid or lipid-like in nature,although alternative liposome bilayer formulations, comprising asurfactant with either a lipid or a cholesterol, exist.

Liposome vesicles may be particularly preferred in that they find manytherapeutic, diagnostic, industrial, and commercial applications. Theyare used to deliver molecules which are not readily soluble in water, orwhen directed timed release is desired. Because of their selectivepermeability to many chemical compounds, liposomes are useful asdelivery vehicles for drugs and biological materials. Thus,lipid-derivatized proteins such as hedgehog can be made multimeric bybeing incorporated into the lipid bilayer of liposome vesicles. Uponreaching the target site, the liposomes may be degraded (for example, byenzymes in the gastro-intestinal tract) or they may fuse with themembranes of cells.

Several methods of preparing vesicles such as liposomes are known. Theproduction of phospholipid vesicles is well known (53). For a generalreview of commonly used methods, see (54). Among the more common ofthese are (1) sonication of a solution containing lipids sometimesfollowed by evaporation/lyophilization and rehydration (see, e.g.Stryer, Biochemistry, pp. 290-291, Freeman & Co., New York, (1988), and(55); (2) homogenization of a lipid solution, sometimes at high pressureor high shearing force (see e.g. U.S. Pat. No. 4,743,449 issued 10 May1988, and U.S. Pat. No. 4,753,788, issued 28 Jun. 1988), (3) hydrationand sometimes sonication of a dried film of vesicle-forming lipidswherein the lipid film is prepared by evaporation of a solution oflipids dissolved in an organic solvent (see e.g. U.S. Pat. No. 4,452,747issued 5 Jun. 1984, U.S. Pat. No. 4,895,719 issued 23 Jan. 1990, andU.S. Pat. No. 4,946,787 issued 7 Aug. 1990), (4) lyophilization orevaporation and rehydration (see e.g. U.S. Pat. No. 4,897,355 issued 30Jan. 1990, EP 267,050 published 5 Nov. 1988, U.S. Pat. No. 4,776,991issued 11 Oct. 1988, EP 172,007 published 19 Feb. 1986, and Australianpatent application AU-A-48713/85 published 24 Apr. 1986), (5) solventinjection or infusion of a lipid solution into an aqueous medium or viceversa (see e.g. (56); U.S. Pat. No. 4,921,757 issued 1 May 1990, U.S.Pat. No. 4,781,871 issued 1 Nov. 1988, WO 87/02396 published 24 Mar.1988, and U.S. Pat. No. 4,895,452 issued 23 Jan. 1990), (6) spray drying(see e.g. Australian patent application AU-A-48713/85 published 24 Apr.1986, and U.S. Pat. No. 4,830,858 issued 16 May 1989), (7) filtration(see e.g. WO 85/01161), (8) reverse-phase evaporation. See e.g. (57);and (9) combinations of the above methods. See e.g. (58) and (59).

Preferred lipids and lipid-like components suitable for use in preparingvesicles include phospholipids, a mixture of phospholipids, polarlipids, neutral lipids, fatty acids, and their derivatives. A preferredlipid has the characteristic that when dispersed alone in water, at atemperature above the lipid transition temperature, they are in a lipidemulsion phase. In certain embodiments, the lipid is a single-aliphaticchain of greater than about 12 carbons and can be either saturated orunsaturated, or substituted in other ways. Suitable lipids include theester, alcohol, and acid forms of the following fatty acids: stearate,oleic acid, linoleic acid, arachidate, arachidonic acid, and othersingle-aliphatic chains acids. Further candidates include the ester,alcohol, and acid forms of the retinols, in particular, retinol andretinoic acid. Other preferred lipids include phosphatidylcholine (PC),phosphatidylglycerol (PG) and their derivatives, created syntheticallyor derived from a variety of natural sources.

In certain embodiments, the vesicle may be stabilized sterically by theincorporation of polyethylene glycol (PEG), or by the PEG headgroups ofa synthetic phospholipid (PEG conjugated to distearoylphosphatidylethanolamine (DSPE), see e.g. (61)). Preferred surfactantsare those with good miscibility such as Tween™, Triton™, sodium dodecylsulfate (SDS), sodium laurel sulfate, or sodium octylglycoside.

Preferred surfactants form micelles when added to aqueous solution abovethe surfactant's phase transition temperature. The surfactants may becomposed of one or more aliphatic chains. These aliphatic chains may besaturated, unsaturated, or substituted in other ways, such as byethoxylation; typically the aliphatic chain contains greater than about12 carbons. Additional suitable surfactants include the following:lauryl-, myristyl-, linoleyl-, or stearyl-sulfobetaine; lauryl-,myristyl-, linoleyl- or stearyl-sarcosine; linoleyl, myristyl-, orcetyl-betaine; lauroamidopropyl-, cocamidopropyl-, linoleamidopropyl-,myristamidopropyl-, palmidopropyl-, or isostearamidopropyl-betaine (e.g.lauroamidopropyl); myristamidopropyl-, palmidopropyl-, orisostearamidopropyl-dimethylamine; sodium methyl cocoyl-, or disodiummethyl oleyl-taurate; and the MONAQUAT series (Mona Industries, Inc.,Paterson, N.J.). See also Example 4.

Preferred sterols and sterol esters suitable for use in preparingmultimeric protein complexes include cholesterol, cholestanol,cholesterol sulfate, and other cholesterol analogs and derivatives. Thefact that a vesicle may comprise many different lipids and detergentsallows great flexibility in engineering a tethered protein-vesiclecomplex with desired properties. For example, one may produce vesiclesthat bind different number of proteins by varying the lipid compositionof the starting materials to create larger vesicles, or by increasingthe percentage of phosphatidylinositol lipids in the vesicle.

VII. Utilities

Generally, the modified proteins described herein are useful fortreating the same medical conditions that can be treated with theunmodified forms of the proteins. However, the hydrophobically-modifiedproteins described herein provide several significant improvements overthe unmodified forms. First, their increased potency enables treatmentwith smaller amounts of protein and over shorter periods of time. Thiswill be important in both systemic and CNS applications. Secondly,replacement of the N-terminal cysteine with a less chemically reactiveamino acid allows for easier production, formulation, and storage of aprotein for clinical use. Thirdly, the pharmacodynamics of a proteinwill be altered by hydrophobic modification and this will allow theproteins to be localized in the vicinity of the site of administration,thus increasing their safety, by minimizing systematic exposure, andtheir effectiveness by increasing their local concentration. Theproteins of the invention are also useful in diagnostic compositions andmethods to detect their corresponding receptors.

As an example of the first point, it has been found that the half-lifeof hedgehog is very short after systemic application and that multipleinjections are required to achieve a robust response to the protein. Thehigher potency of the modified forms and the possibility of formulationin liposomes provides a means of achieving a response with fewertreatments. For CNS applications, the higher potency provides a means tosupply an adequate amount of protein in the small volumes required fordirect injection into the CNS.

The importance of the second point is illustrated by the fact that wehave found that the N-terminal cysteine of hedgehog is highlysusceptible to chemical attack, either to form other chemical adducts orto oxidatively-dimerize with another hedgehog protein. To prevent this,special buffers and procedures are used during purification, anddithiothreitol is used in the final formulation. These precautionsnecessitate careful evaluation of the effects of the formulation bufferin animal models.

As an example of the third point, the more limited the range over whicha protein diffuses away from the site of administration, the higher thelocal concentration. This higher local concentration may therefore allowmore specific clinical responses during the treatment of neurologicaldisorders after direct injection into the desired region of the brain orspinal cord.

Similarly, the modified proteins can be administered locally to the siteof bone fractures to help heal these fractures, in the gonads to treatfertility disorders, intraocularly to treat eye disorders, and under theskin to treat dermatological conditions, and to stimulate local hairgrowth. Localization of the hydrophobically-modified proteins to thesite of administration therefore reduces possibly undesirable systemicexposure to other tissues and organs.

For therapeutic use, hydrophobically-modified proteins of the inventionare placed into pharmaceutically acceptable, sterile, isotonicformulations and optionally are administered by standard means wellknown in the field. The formulation is preferably liquid or may belyophilized powder. It is envisioned that a therapeutic administrationof, for instance, a multimeric protein complex may comprise liposomesincorporating the derivatized proteins described herein.

It will be appreciated by persons having ordinary skill in the art thatthe particular administration, dosage, and clinical applications of ahydrophobically-modified protein of the invention will vary dependingupon the particular protein and its biological activity.

As but one example of the application of the proteins of this inventionin a therapeutic context, therapeutic hydrophobically-modified hedgehogproteins can be administered to patients suffering from a variety ofneurological conditions. The ability of hedgehog protein to regulateneuronal differentiation during development of the nervous system andalso presumably in the adult state indicates thathydrophobically-modified hedgehog can reasonably be expected tofacilitate control of adult neurons with regard to maintenance,functional performance, and aging of normal cells; repair andregeneration processes in lesioned cells; and prevention of degenerationand premature death which results from loss of differentiation incertain pathological conditions. In light of this, the presenthydrophobically-modified hedgehog compositions, by treatment with alocal infusion can prevent and/or reduce the severity of neurologicalconditions deriving from: (i) acute, subacute, or chronic injury to thenervous system, including traumatic injury, chemical injury, vesselinjury, and deficits (such as the ischemia from stroke), together withinfectious and tumor-induced injury; (ii) aging of the nervous systemincluding Alzheimer's disease; (iii) chronic neurodegenerative diseasesof the nervous system, including Parkinson's disease, Huntington'schorea, amylotrophic lateral sclerosis and the like; and (iv) chronicimmunological diseases of the nervous system, including multiplesclerosis. The hydrophobically-modifed protein may also be injected intothe cerebrospinal fluid, e.g., in order to address deficiencies of braincells, or into the lymph system or blood stream as required to targetother tissue or organ system-specific disorders.

Hedgehog compositions of the invention may be used to rescue, forexample, various neurons from lesion-induced death as well as guidingreprojection of these neurons after such damage. Such damage can beattributed to conditions that include, but are not limited to, CNStrauma infarction, infection, metabolic disease, nutritional deficiency,and toxic agents (such as cisplatin treatment). Certain hedgehogproteins cause neoplastic or hyperplastic transformed cells to becomeeither post-mitotic or apoptotic. Such compositions may, therefore, beof use in the treatment of, for instance, malignant gliomas,medulloblastomas and neuroectodermal tumors.

The proteins may also be linked to detectable markers, such asfluoroscopically or radiographically opaque substances, and administeredto a subject to allow imaging of tissues which express hedgehogreceptors. The proteins may also be bound to substances, such ashorseradish peroxidase, which can be used as immunocytochemical stainsto allow visualization of areas of hedgehog ligand-positive cells onhistological sections. Hydrophobically-modified proteins of theinvention, either alone or as multivalent protein complexes, can be usedto specifically target medical therapies against cancers and tumorswhich express the receptor for the protein. Such materials can be mademore effective as cancer therapeutics by using them as delivery vehiclesfor antineoplastic drugs, toxins, and cytocidal radionuclides, such asyttrium 90.

A toxin may also be conjugated to hydrophobically-modified hedgehog (orvesicle-containing multivalent complexes thereof) to selectively targetand kill hedgehog-responsive cells, such as a tumor expressing hedgehogreceptor(s). Other toxins are equally useful, as known to those of skillin the art. Such toxins include, but are not limited to, Pseudomonasexotoxin, Diphtheria toxin, and saporin. This approach should provesuccessful because hedgehog receptor(s) are expressed in a very limitednumber of tissues. Another approach to such medical therapies is to useradioisotope labeled, hydrophobically-modified protein (or multivalentcomplexes thereof). Such radiolabeled compounds will preferentiallytarget radioactivity to sites in cells expressing the proteinreceptor(s), sparing normal tissues. Depending on the radioisotopeemployed, the radiation emitted from a radiolabeled protein bound to atumor cell may also kill nearby malignant tumor cells that do notexpress the protein receptor. A variety of radionuclides may be used.Radio-iodine (for example, ¹³¹I) has been successful when employed withmonoclonal antibodies against CD20 present on B-cell lymphomas (63).

The protein compositions to be used in therapy will be formulated anddosages established in a fashion consistent with good medical practicetaking into account the disorder to be treated, the condition of theindividual patient, the site of delivery of the isolated polypeptide,the method of administration, and other factors known to practitioners.The therapeutic may be prepared for administration by mixing a protein,a protein-containing vesicle, or a derivatized complex at the desireddegree of purity with physiologically acceptable carriers (i.e. carrierswhich are nontoxic to recipients at the dosages and concentrationsemployed).

It is envisioned that local delivery to the site will be the primaryroute for therapeutic administration of the proteins of this invention.Intravenous delivery, or delivery through catheter or other surgicaltubing may also be envisioned. Alternative routes include tablets andthe like, commercially available nebulizers for liquid formulations, andinhalation of lyophilized or aerosolized formulations. Liquidformulations may be utilized after reconstitution from powderformulations.

The dose administered will be dependent upon the properties of thevesicle and protein employed, e.g. its binding activity and in vivoplasma half-life, the concentration of the vesicle and protein in theformulation, the administration route, the site and rate of dosage, theclinical tolerance of the patient involved, the pathological conditionafflicting the patient and the like, as is well known within the skillof the physician. Generally, doses of from about 5×10⁻⁷ to 5×10⁻⁹ Molarof protein per patient per administration are preferred, although thedosage will depend on the nature of the protein. Different dosages maybe utilized during a series of sequential administrations.

The invention is also directed towards a pharmaceutical formulationwhich includes a hedgehog protein modified according to the invention incombination with a pharmaceutically acceptable carrier. In oneembodiment, the formulation also includes vesicles.

The hydrophobically-modified hedgehog proteins of the invention are alsouseful in gene therapy methods.

For neurodegenerative disorders, several animal models are availablethat are believed to have some clinical predicative value. ForParkinson's disease, models involve the protection, or the recovery inrodents or primates in which the nigral-striatal dopaminergic pathway isdamaged either by the systemic administration of MPTP or the local(intracranial) administration of 6-hydroxydopamine [6-OHDA], twoselective dopaminergic toxins. Specific models are: MPTP-treated mousemodel (64); MPTP-treated primate (marmoset or Rhesus) model (65), andthe unilateral 6-OHDA lesion rat model (66). For ALS, (Amyotrophiclateral sclerosis) models involve treatment of several mice strains thatshow spontaneous motor neuron degeneration, including the wobbler (67)and pmn mice (68), and of transgenic mice expressing the human mutatedsuperoxidase dismutase (hSOD) gene that has been linked to familial ALS(69). For spinal cord injury, the most common models involve contusioninjury to rats, either through a calibrated weight drop, or fluid(hydrodynamic) injury (70). For Huntington's. models involve protectionfrom excitotoxin (NMDA, quinolinic acid, kainic acid, 3-nitro-propionicacid, APMA) lesion to the striatum in rats (71, 72). Recently, a modelof transgenic mice overexpressing the human trinucleotide expandedrepeat in the huntingtin gene has also been described (73). For multiplesclerosis, EAE in mice and rats is induced by immunization with MBP(myelin basic protein), or passive transfer of T cells activated withMBP (74). For Alzheimer's, a relevant murine model is a determination ofprotection against lesion of the fimbria-fornix in rats (septal lesion),the main nerve bundle supplying the cholinergic innervation of thehippocampus (75), as well as use of transgenic mice overexpressing thehuman beta-amyloid gene. For peripheral neuropathies a relevant model isprotection against loss of peripheral nerve conductance caused bychemtherapeutic agents such as taxol, vincristine, and cisplatin in miceand rats (76).

This invention will now be described more fully with reference to thefollowing, non-limiting, Examples.

EXAMPLE 1 Human Sonic Hedgehog is Lipid-Modified when Expressed inInsect Cells

A. Expression of Human Sonic Hedgehog

The cDNA for fill-length human Sonic hedgehog (Shh) was provided as a1.6 kb EcoRI fragment subcloned into pBluescript SK⁺ (20) (a gift ofDavid Bumcrot from Ontogeny, Inc., Cambridge Mass.). The 5′ and 3′ NotIsites immediately flanking the Shh open reading frame were added byunique site elimination mutagenesis using a Pharmacia kit following themanufacturer's recommended protocol. The 1.4 kb NotI fragment carryingthe full-length Shh cDNA was then subcloned into the insect expressionvector, pFastBac (Life Technologies, Inc.). Recombinant baculovirus wasgenerated using the procedures supplied by Life Technologies, Inc. Theresulting virus was used to create a high titer virus stock. Methodsused for production and purification of Shh are described below. Thepresence of membrane-associated Shh was examined by FACS and by Westernblot analysis. Peak expression occurred 48 h post-infection. For Westernblot analysis, supernatants and cell lysates from Shh-infected oruninfected cells were subjected to SDS-PAGE on a 10-20% gradient gelunder reducing conditions, transferred electrophoretically tonitrocellulose, and the Shh detected with a rabbit polyclonal antiserumraised against an N-terminal Shh 15-mer peptide-keyhole limpethemocyanin conjugate. The cell lysates were made by incubating the cellsfor 5 min at 25° C. in 20 mM Na₂HPO₄ pH 6.5, 1% Nonidet P40 and 150 mMNaCl or 20 mM Tris-HCl pH 8.0, 50 mM NaCl, 0.5% Nonidet P40 and 0.5%sodium deoxycholate and then pelleting particulates at 13,000 rpm for 10min at 4° C. in an Eppendorf centrifuge.

B. Purification of Membrane-Tethered Human Sonic Hedgehog

The membrane-tethered form of Shh was produced in High Five™ insectcells (Invitrogen) using the recombinant baculovirus encodingfull-length Shh discussed above. High Five™ cells were grown at 28° C.in sf900 II serum free medium (Life Technologies, Inc.) in a 10 Lbioreactor controlled for oxygen. The cells were infected in late logphase at ca. 2×10⁶ cells/mL with virus at a MOI of 3 and harvested 48 hafter infection (cell viability at the time of harvest was >50%). Thecells were collected by centrifugation and washed in 10 mM Na₂HPO₄ pH6.5, 150 mM NaCl pH plus 0.5 mM PMSF. The resulting cell pellet (150 gwet weight) was suspended in 1.2 L of 10 mM Na₂HPO₄ pH 6.5, 150 mM NaCl,0.5 mM PMSF, 5 μM pepstatin A, 10 μg/mL leupeptin, and 2 μg/mL E64, and120 mL of a 10% solution of Triton X-100 was then added.

After a 30 min incubation on ice, particulates were removed bycentrifugation (1500×g, 10 min). All subsequent steps were performed at4-6° C. The pH of the supernatant was adjusted to 5.0 with a stocksolution of 0.5 M MES pH 5.0 (50 mM final) and loaded onto a 150 mlSP-Sepharose Fast Flow column (Pharmacia). The column was washed with300 mL of 5 mM Na₂HPO₄ pH 5.5, 150 mM NaCl, 0.5 mM PMSF, 0.1% NonidetP-40, then with 200 mL of 5 mM Na₂HPO₄ pH 5.5, 300 mM NaCl, 0.1% NonidetP-40, and bound hedgehog eluted with 5 mM Na₂HPO₄ pH 5.5, 800 mM NaCl,0.1% Nonidet P-40.

The Shh was next subjected to immunoaffinity chromatography on5E1-Sepharose resin that was prepared by conjugating 4 mg of antibodyper mL of CNBr activated Sepharose 4B resin. The SP-Sepharose elutionpool was diluted with two volumes of 50 mM HEPES pH 7.5 and batch loadedonto the 5E1 resin (1 h). The resin was collected in a column, washedwith 10 column volumes of PBS containing 0.1% hydrogenated Triton X-100(Calbiochem), and eluted with 25 mM NaH₂PO₄ pH 3.0, 200 mM NaCl, 0.1%hydrogenated Triton X-100. The elution fractions were neutralized with0.1 volume of 1M HEPES pH 7.5 and analyzed for total protein contentfrom absorbance measurements at 240-340 nm and for purity by SDS-PAGE.Fractions were stored at −70° C.

Peak fractions from three affinity steps were pooled, diluted with 1.3volumes of 50 mM HEPES pH 7.5, 0.2% hydrogenated Triton X-100 and againbatch loaded onto the 5E1 resin. The resin was collected in a column,washed with three column volumes of PBS pH 7.2, 1% octylglucoside (USBiochemical Corp.), and eluted with 25 mM NaH₂PO₄ pH 3.0, 200 mM NaCl,1% octylglucoside. The elution fractions were neutralized and analyzedas described above, pooled, filtered through a 0.2 micron filter,aliquoted, and stored at −70° C.

When full-length human sonic hedgehog (Shh) was expressed in High Five™insect cells, over 95% of the N-terminal fragment was detected byWestern blotting in a form that was cell-associated. By SDS-PAGE, thepurified protein migrated as a single sharp band with apparent mass of20 kDa (FIG. 1, lane c). The protein migrated faster by about 0.5 kDathan a soluble version of the protein that had been produced in E. coli(FIG. 1, lanes b-d), consistent with data published previously (19).Similarly as described (19), the soluble and membrane-bound Shh proteinswere also readily distinguishable by reverse phase HPLC where thetethered form eluted later in the acetonitrile gradient. Theconcentration of acetonitrile needed for elution of the membrane-boundform was 60% versus only 45% with the soluble form, indicating asignificant increase in the hydrophobicity of the protein.

C. Mass Spectrometry Analysis of Membrane-Tethered Human Sonic Hedgehog

Aliquots of Shh were subjected to reverse phase HPLC on a C₄ column(Vydac, Cat. No. 214TP104, column dimensions 4.6 mm internaldiameter×250 mm) at ambient temperature. Bound components were elutedwith a 30 min 0-80% gradient of acetonitrile in 0.1% trifluoroaceticacid at a flow rate of 1.4 mL/min. The column effluent was monitored at280 nm and 0.5 min fractions were collected. 25 μL aliquots of fractionscontaining protein were dried in a Speed Vac concentrator, dissolved inelectrophoresis sample buffer, and analyzed by SDS-PAGE.Hedgehog-containing fractions were pooled, concentrated 4-fold in aSpeed Vac concentrator and the protein content assayed by absorbance at280 nm using an extinction coefficient of 1.33 for a 1 mg/mL solution ofShh. Samples were subjected to ESI-MS on a Micromass Quattro II triplequadrupole mass spectrometer, equipped with an electrospray ion source.A volume of 6 μL of HPLC-purified hedgehog was infused directly into theion source at a rate of 10 μL/min using 50% water, 50% acetonitrile,0.1% formic acid as the solvent in the syringe pump. Scans were acquiredthroughout the sample infusion. All electrospray mass spectral data wereacquired and stored in profile mode and were processed using theMicromass MassLynx data system.

Peptides from an endoproteinase Lys-C digest of pyridylethylated-Shhwere analyzed by reverse phase HPLC in line with the Micromass QuattroII triple quadrupole mass spectrometer. The digest was separated on aReliasil C₁₈ column using a Michrom™ ultrafast Microprotein Analyzersystem at a flow rate of 50 μL/min with a 5-85% acetonitrile gradient in0.05% trifluoroacetic acid. Scans were acquired from m/z 400-2000throughout the run and processed as described above.

Sequencing of the N-terminal peptide from tethered Shh was performed byPost Source Decay (PSD)-measurement on a Voyager-DE™ STR (PerSeptiveBiosystems, Framingham, Mass.) time-of-flight (TOF) mass spectrometerusing α-cyano-4-hydroxycinnamic acid as the matrix (22,23). Exactly 0.5μL of HPLC-purified endoproteinase Lys-C peptide was mixed with 0.5 μLof matrix on the target plate. To cover the entire spectrum of fragmentions, the mirror voltages were decreased from 20 to 1.2 kv in 11 steps.

Electrospray ionization mass spectrometry data for the soluble andmembrane-bound forms of Shh showed primary species with masses of 19560and 20167 Da, respectively (FIG. 2). The measured mass of 19560 Damatches the predicted mass for Shh starting with Cys-1 and terminatingwith Gly-174 (calc. mass of 19560.02 Da). In contrast, the 20167 Da massdid not agree with any available prediction nor could the difference inthe masses of the tethered and soluble forms, 607 Da, be accounted forby any known modification or by aberrant proteolytic processing.Previously, Porter et al. (18) demonstrated that Drosophila hedgehogcontained a cholesterol moiety and thus it was possible that the massdifference in the human system was due, at least in part, to cholesterol(calculated mass for esterified cholesterol is 368.65 Da). The presenceof a minor component in the mass spectrum of tethered Shh at 19796 Da,which differs from the primary peak by 371 Da, supported this notion.

Further evidence for cholesterol was obtained by treating the tetheredShh with a mild alkali under conditions that can break the cholesterollinkage without disrupting peptide bonds (18), and then reanalyzing thereaction products by mass spectrometry (MS). Briefly, insectcell-derived Shh was treated with 50 mM KOH, 95% methanol for 1 h atambient temperature and then analyzed by ESI-MS or digested withendoproteinase Lys-C and subjected to LC (liquid chromatography)-MS onthe Micromass Quattro II triple quadrupole mass spectrometer. Forsamples subjected to LC-MS, the proteins were first treated with4-vinylpyridine. Base treatment shifted the mass by 387 Da, which isconsistent with the loss of cholesterol plus water (see Table 3). Themass of soluble Shh was not affected by base treatment. Together, theseobservations suggested that the membrane-tethered human Shh containedtwo modifications, a cholesterol and a second moiety with a mass of 236Da. The similarity in mass between this value and the mass of an addedpalmitoyl group (238 Da) suggested that the protein might bepalmitoylated. More accurate estimates of the mass, discussed below,revealed a correlation within 0.1 Da of the predicted mass of apalmitoyl moiety. TABLE 3 Characterization of tethered Shh by MS.Calculated mass values were determined using average residue masses inpart a and monoisotopic protonated masses in part b. Mass (Da) ProteinCalculated Measured a. KOH-treated Shh no tether (−treatment) 19560.0219560 no tether (+treatment) 19560.02 19561 tethered (−treatment)20167.14 20167 tethered (+treatment) 19798.49 19780 b. N-terminalendoproteinase Lys-C peptide (MH⁺)* no tether 983.49 983.50 tethered1221.72 1221.79*All mass values for peptides described herein are protonated masses.

Subsequently, we determined that tethered Shh could be fractionated intosubspecies by HPLC with a modified elution gradient and we developed asimple HPLC assay for quantifying the various forms. Results from theseanalyses are shown in FIG. 3. In this assay, the unmodified Shh elutesfirst (peak 1), then cholesterol-modified Shh elutes (peak 2), andfinally product containing both cholesterol and palmitic acid-modifiedShh elutes (peak 3). The complex shape of peak 3 reflects the presenceof a modified form of the palmitoyl group that was identified throughsequencing by MALDI PSD measurement. The variant was 2 Da smaller thanpredicted and may therefore contain an unsaturated bond (data notshown).

D. Localization of the Palmitic Acid Modification Within the Human SonicHedgehog Sequence

The site of palmitoylation within the human sequence was identifiedusing a combination of peptide mapping and sequence analysis. FIG. 4Bshows results from a peptide mapping analysis of the soluble proteinwith an LC-MS readout. Mass data accounting for over 98% of the solubleShh sequence could be accounted for from the analysis. The peak notedwith an asterisk corresponds to the N-terminal peptide (residues 1-9plus 4-vinylpyridine, observed mass 983.50 Da, calculated mass 983.49Da; Table 3). In the corresponding analysis of the tethered product(FIG. 4A), this peptide was missing and instead a more hydrophobicpeptide with mass of 1221.79 Da was observed (noted with asterisk).

The 1221.79 Da moiety is consistent with the presence of a modified formof the N-terminal peptide, i.e. 983.49 Da for the peptide component plus238.23 Da. The 1221.79 Da peptide was next subjected to sequenceanalysis by MALDI PSD measurement. The resulting PSD spectrum is shownin FIG. 5. Ions corresponding to b1, b2, b4, b5, b8+H₂O, y8, y7, y5, y4,y3, y2, and y1 fragments were detected which confirmed the sequence. Inaddition, the b1 and b2 ions indicated that the pyridylethylated Cys-1adduct was palmitoylated. Only ions containing Cys-1, contained theadded 238.23 Da mass.

Since cysteine is a normal site of palmitoylation for proteins in vivo,it was not surprising to find the novel adduct attached to theN-terminal cysteine. However, two pieces of evidence suggested that thelipid was attached to the amino group on the cysteine and not the thiol.First, in the peptide mapping study, we used 4-vinylpyridine as aspectroscopic tag to monitor free thiol groups (27). Pyridylethylationis highly specific for cysteine thiols and adds a 105 Da adduct that canbe detected by MS. The observed Cys-1-containing fragments in the PSDspectrum contained both palmitoyl and pyridylethyl modifications,implying the presence of a free thiol group. Second, the tethered Shhwas subjected to automated N-terminal Edman sequencing and no sequencewas obtained, suggesting blockage at the N-terminal α-amine. Bycontrast, the corresponding soluble form of Shh can be sequencedreadily.

EXAMPLE 2 Human Sonic Hedgehog can be Modified with Palmitic Acid in aCell-Free System

Soluble Shh was labeled with ³H-palmitic acid in a cell-free systemusing a modified version of a published procedure (24). A crudemicrosomal fraction from rat liver was prepared by subjecting a liverhomogenate to sequential centrifugation at 3000×g for 10 min, 9000×g for20 min, and 100,000×g for 30 min. The 100,000×g pellet was suspended in10 mM HEPES pH 7.4, 10% sucrose and again centrifuged at 100,000×g for20 min. The final pellet (derived from 10 g of liver) was suspended in 3mL of 20 mM Tris-HCl pH 7.4, 150 mM NaCl, 1 mM EDTA, 10 μg/mL leupeptin,0.15% Triton X-100, aliquoted, and stored at −70° C. Reactionscontaining 3 μg Shh, 1 μL of rat microsomes, 50 ng/mL Coenzyme A(Sigma), 0.3 mM ATP, 20 mM Tris-HCl pH 7.4, 150 mM NaCl, 1 mM EDTA, 10μg/mL leupeptin, and 0.5 μCi-[9,10-³H]-palmitic acid (50 Ci/mmol; NewEngland Nuclear) were performed at room temperature for 1 h. Reactionswere stopped with reducing electrophoresis sample buffer, subjected toSDS-PAGE on a 10-20% gradient gel, and visualized by fluorography.

As shown in FIG. 1 (lane e), Shh is readily labeled with the radioactivetracer. None of the ca. hundred other proteins in the reaction mixturewere labeled (see the corresponding Coomassie blue-stained gel profilein lane j), indicating a high degree of specificity of thepalmitoylation reaction. As further evidence for the specificity of thepalmitoylation reaction, we tested two Shh variants in which the site ofpalmitoylation had been eliminated. FIG. 1 (lane f) shows results fromthe analysis of a truncated form of soluble Shh that was lacking thefirst 10 amino acid residues of the mature sequence) and lane g, of amutant form of Shh containing, at its N-terminal end, a single Cys-1 toSer point mutation. Neither of the variants were labeled.

The significance of the N-terminal cysteine as the site of lipidderivatization is highlighted by the fact that wild type soluble Shh isreadily labeled while the N-terminal cysteine to serine mutant is not.The inability to label the N-terminal serine mutant argues against asimple reaction mechanism where the palmitoyl moiety is directlyattached to the N-terminal α-amine since under the test conditions theserine should have substituted for the cysteine.

We also tested the role of the free N-terminus using a form of solubleShh with an N-terminal histidine (His)-tag extension. The soluble humanShh used in these studies had been produced initially as a His-taggedfusion protein with an enterokinase cleavage site at the junction of themature sequence and was then processed with enterokinase to remove theHis tag. The His-tagged Shh was not palmitoylated despite the presenceof the free thiol group of the cysteine (See FIG. 1, lane i). While wecannot rule out the possibility that the N-terminal extension stericallyinhibits palmitoylation from occurring, Cys-1 is at the P1′ position ofthe enterokinase cleavage site and is readily accessible to enzymaticprocessing. Thus it appears that both the thiol and α-amine of Cys-1contribute to the palmitoylation reaction. Since all knownpalmitoylation reactions target the side chains of Cys, Ser, or Thrresidues, we infer that the modification on hedgehog starts with theformation of a thioester intermediate, and that the palmitoyl moiety isthen transferred to the N-terminus through the formation of a cyclicintermediate. This hypothesis was confirmed during studies of themodification of human Sonic hedgehog using palmityol Coenzyme A (SeeExample 8).

EXAMPLE 3 Demonstration of Increased Potency of Naturally OcurringFatty-Acylated Human Sonic Hedgehog in a Cell-Based (C3H10T1/2) Assay

Shh was tested for function in a cell-based assay measuring alkalinephosphatase induction in C3H10T1/2 cells (25) with a 5 day readout. Theassay was preformed in a 96-well format. Samples were run in duplicate.For tethered Shh (100 μg/mL), the samples were first diluted 200-foldwith normal growth medium then subjected to serial 2-fold dilutions downthe plates. Wells were normalized for potential effects of the addedoctylglucoside by including 0.005% octylglucoside in the culture medium.Blocking studies using the neutralizing murine mAb 5E1 (26) wereperformed by mixing Shh with serial dilutions of the antibody for 30 minat ambient temperature in culture medium prior to adding the testsamples to the plates.

In this assay, soluble human Shh produces a dose-dependent response withan IC₅₀ of 1 μg/mL and a maximal signal at 3 μg/mL (FIG. 6A). Tetheredhuman Shh, with a cholesterol attached at the C-terminus and a palmitoylgroup at the N-terminus, similarly produced a dose-dependent response inthe assay but with an IC₅₀ of 0.03 μg/mL and a maximal signal at 0.1μg/mL, indicating that it was about 30 times as potent as soluble Shh.To verify that the observed activity was hedgehog specific, we testedwhether the activity could be inhibited with the anti-hedgehogneutralizing mAb 5E1. Both soluble and tethered Shh were inhibited by5E1 treatment (FIG. 6B). Inhibition of the tethered Shh required a tenthas much 5E1 consistent with its increased activity in the assay.

Tethered Shh was tested in a receptor binding assay, monitoring itsability to bind patched, using a modified version of a recentlypublished assay (10). The tethered Shh showed dose-dependent binding tocells expressing patched with an apparent IC₅₀ of 400 ng/mL (FIG. 7). Inthe same assay, soluble Shh bound to patched with an apparent IC₅₀ of150 ng/mL, indicating that the tethered form bound only slightly lesstightly to its receptor.

EXAMPLE 4 Analysis of Tethered Human Sonic Hedgehog After ReconstitutionInto Liposomes

This example illustrates that reconstitution experiments into positivelyand negatively charged liposomes by detergent dilution over a wide rangeof lipid:protein ratios (w/w) from 1:1 to 100:1 had no effect ontethered Shh activity in the C3H10T1/2 assay.

Reconstitution into phospholipid-containing liposomes provides a usefulformulation for lipid-containing proteins because it allows alipid-containing protein to exist in a near normal setting. To testwhether such a formulation was viable for tethered Shh we utilized adetergent dilution method to incorporate the protein into a liposome(60), where preformed liposomes are mixed with octylglucoside and theprotein of interest, and then the detergent is diluted below itscritical micelle concentration, thus driving the reconstitution. Whileany of a large number of pure or lipid mixtures can be utilized, weselected two commercially available mixtures as models; a negativelycharged liposome kit containing egg L-α-phosphatidylcholine, dicetylphosphate, and cholesterol (Cat. No. L-4262; Sigma, St. Louis, Mo.), anda positively charged liposome kit consisting of egg phosphatidylcholine, stearlyamine, and cholesterol (Cat. No L-4137, Sigma).

Briefly, the lipids were transferred into a Pyrex tube, dried under astream of nitrogen, and residual solvent removed by lyophilization. Thelipid was suspended in 10 mM HEPES pH 7.5, 100 mM NaCl, 2.0%octylglucoside, vortexed, and sonicated until the suspension had turnedopalescent in appearance. The lipid was then filtered through a 0.2micron filter. Aliquots of tethered Shh, from baculovirus-infected HighFive™ insect cells, in octylglucoside were treated with a 400-, 1000-,5000-, and a 20000-fold excess of lipid (w/w) and after a 15 minpreincubation the samples were diluted and assayed for activity in theC3H10T1/2 assay.

Neither the positive nor the negative liposome treatment had any affecton the activity of the hedgehog indicating that a lipid carrier was aviable formulation. To verify that the hedgehog indeed had becomereconstituted, parallel samples were subjected to centrifugation underconditions where the tethered Shh would normally pellet and theliposomes would float to the surface of the sample. Under theseconditions the tethered Shh floated to the surface, indicating thatreconstitution had occurred.

EXAMPLE 5 Characterization of Membrane-Tethered Human Sonic Hedgehogfrom Mammalian (EBNA-293) Cells

In order to assess whether palmitoylation was a general modificationpathway for Sonic hedgehog or whether it was specific to insect cellproduction, the protein was also produced in a mammalian system inEBNA-293 cells. For expression of full-length Shh in mammalian cells,the 1.4 kb NotI fragment containing full-length Shh (See Example 1) wascloned into a derivative of the vector, CH269 pCEP4 (Invitrogen, SanDiego, Calif. (21)). The construct was transfected into EBNA-293 cellsusing lipofectamine (Life Technologies, Inc.) and the cells wereharvested 48 h post-transfection. The expression of surface Shh wasverified by FACS and by Western blot analysis.

Tethered Shh from EBNA-293 cells was fractionated by reverse phase HPLCon a narrow bore C₄ column (See FIG. 3). Peaks were analyzed by ESI-MS(parts a and b of Table 4) or by MALDI-TOF MS on a Finnigan LaserMatmass spectrometer using α-cyano4-hydroxycinnamic acid as the matrix(part c of Table 4). By SDS-PAGE, the protein migrated slightly fasterthan soluble Shh, it was retarded on the C₄ column in the reverse phaseHPLC analysis, and, by mass spectrometry, it contained an ioncorresponding to the palmitic acid plus cholesterol modification.However, unlike the insect cell-derived product where over 80% of theproduct contained both the palmitic acid and cholesterol modification,the HPLC elution profile and data from mass spectrometry revealed thatmost of the mammalian cell-derived protein lacked the palmitoyl moiety(see Table 4 and FIG. 3C). That is, in peak 2 from EBNA-293 cells theratio of clipped (des-1-10) versus intact protein by MS signal was 50%whereas for peak 1 only about 10% of the Shh was clipped. Interestingly,both the insect cell and mammalian cell-derived products showedcomparable activity in the C3H10T1/2 assay suggesting that both thecholesterol, and the cholesterol plus palmitic acid modifications arefunctional. Whether the second lipid attachment site is used simply tofurther stabilize the association of the protein with membrane orwhether it plays a more active role and affects its conformation orprotein-protein contacts remains to be determined.

Fatty acid derivatization of proteins is a common post translationalmodification that occurs late in the maturation processes (28,29). Forcysteine derivatives, the process is dynamic involving separate enzymesthat add and remove the modification on the sulfhydryl group. The mostcommon functions of such derivatization (e.g., palmitoylation) are toalter the physico-chemical properties of the protein, i.e., to target aprotein to its site of function, to promote protein-proteininteractions, and to mediate protein-membrane interactions (30). Forhedgehog, while the difference in the extent of palmitoylation in theinsect and mammalian cell-derived preparations (80% in insect cellsversus 30% in mammalian cells) was surprising, we do not know if it isbiologically significant or whether it simply reflects differences inthe cellular machinery of the two test systems for adding and removingpalmitic acid. The difference in the extent of modification in theinsect and mammalian cells is unlikely to be species related sincetethered Drosophila hedgehog that was produced in insect cells lackedpalmitic acid (19) despite having the identical N-terminal sequence.TABLE 4 Mass spectrometry analysis of EBNA-293-derived tethered humanSonic hedgehog. Mass (Da) Protein Calculated Measured a. bacterialexpressed (no tether) 19560.02 19560 b. baculovirus expressed(tethered) + palmitic acid 19798.49 19796 + palmitic acid/cholesterol20167.14 20168 c. EBNA-293 cell expressed (tethered) peak 1 (9% of totalhedgehog) no tether 19560.02 19581 no tether (des 1-9) 18700.02 18712peak 2 (61% of total hedgehog) + cholesterol 19928.67 19934 +cholesterol (des 1-10) 18912.48 18889 peak 3 (30% of total hedgehog) +palmitoyl/cholesterol 20167.14 20174

EXAMPLE 6 Lipid Modifications of Rat Sonic Hedgehog

This Example illustrates that a variety of lipids become linked to asoluble version of rat Sonic hedgehog when the rat Shh gene encodingresidues 1-174 is expressed in High Five™ insect cells, essentially asfor the full-length human Shh described in Example 1. The lipidmodification renders this fraction membrane-associated. The N-terminalfragment (residues 1-174 of unprocessed rat Sonic hedgehog) differs byonly 2 amino acid residues to that of the N-terminal fragment of humanSonic hedgehog. In the rat Sonic hedgehog N-terminal fragment, threoninereplaces serine at position 44, and aspartic acid replaces glycine atposition 173. When rat Sonic hedgehog lacking the autoprocessing domainis expressed in the High-Five™ insect cell/baculovirus expressionsystem, the majority of the protein is secreted into the culture mediumsince it lacks the ability to attach a cholesterol moiety to theC-terminus. This soluble form has a specific biological activity(measured by the C3H10T1/2 alkaline phosphatase induction assay ofExample 3) that was similar to that of the soluble, N-terminal fragmentof human Sonic hedgehog expressed and purified from E. coli.

However, a small fraction of the total protein remains associated withthe insect cells. The cell-associated rat Sonic hedgehog protein waspurified essentially as described in Example 1, and was found to besignificantly more active in the alkaline phosphatase assay (data notpresented) than the soluble, N-terminal fragments of either human or ratSonic hedgehog purified from E. coli and the High-Five™ insectcell/baculovirus expression system, respectively. Subsequent analyses ofthe rat Sonic hedgehog N-terminal fragments by HPLC and electrospraymass spectrometry (as described in Example 1) suggests that the proteinis lipid-modified and that there was more than one type of lipidmodification. Supporting evidence includes the following observations:

1. The cell-associated forms elute later than the soluble, N-terminalfragments of human and rat sonic hedgehog from a C₄ reverse phase HPLCcolumn (Vydac catalog number 214TP104) developed with a linear 30 min0-70% acetonitrile gradient in 0.1% trifluoroacetic acid;

2. The masses of the cell-associated forms are consistent with thatexpected for the lipid-modified proteins, as shown in Table 5. TABLE 5Masses of various lipid-modified forms of rat Sonic hedgehog. ExpectedObserved Protein Adduct Mass*(MH⁺) Mass (MH⁺) unmodified none 19,632.0819,632 myristoyl- CH₃(CH₂)₁₂CO— 19,842.50 19,841 palmitoyl-CH₃(CH₂)₁₄CO— 19,870.55 19,868 stearol- CH₃(CH₂)₁₆CO— 19,898.60 19,896arachidoyl- CH₃(CH₂)₁₈CO— 19,926.66 19,925*Average masses were used in calculating the expected masses

The location of the lipid moiety was determined using a combination ofsequence analysis and peptide mapping. Automated N-terminal Edmansequencing of the lipid-modified forms indicated that the N-terminus wasblocked, suggesting that the lipid was attached to the α-amine of theN-terminal cysteine. Endo-Lys-C peptide mapping, MALDI-TOF massspectrometry and MALDI PSD analysis (as described in Example 1) of the4-vinylpyridine alkylated lipid-modified forms, were used to confirm thelocation of the lipid modifications and to determine their exact masses.

The masses of the N-terminal peptides (residues 1-9 inclusive plus4-vinylpyridine attached to the thiol side chain of the N-terminalcysteine) carrying the lipid modifications were consistent within 0.1 Dawith that expected for the lipid-modified peptides as shown in Table 6.TABLE 6 Masses of the N-terminal peptides iosolated from variouslipid-modified forms of rat Sonic hedgehog. Expected Observed ProteinAdduct Mass* (MH⁺) Mass (MH⁺) myristoyl- CH₃(CH₂)₁₂CO— 1193.69 1193.76palmitoyl- CH₃(CH₂)₁₄CO— 1221.72 1221.65 stearoyl- CH₃(CH₂)₁₆CO— 1249.751249.71*Monoisotopic masses were used in calculating the expected masses

In addition to the lipid-modified peptides shown in Table 6, peptideswith masses of 1191.74, 1219.84 and 1247.82 were also detected. Thesemasses are consistent with unsaturated forms of myristate, palmitate andstearate, respectively, although the position of the double bond in thealkyl chain was not determined. These observations indicate that bothsaturated and unsaturated fatty acids can be attached covalently to theN-terminal cysteine. For both the saturated and unsaturatedlipid-modified peptides, MALDI PSD analysis as described in Example 1confirmed that the lipids were attached covalently to the N-terminalcysteine residue.

EXAMPLE 7 Lipid Modification of Indian Hedgehog

To assess whether the palmitoylation reaction was unique to human Shh orwhether it might occur on other hedgehog proteins, we tested whetherhuman Indian hedgehog (expressed in E. coli as a His-tagged fusionprotein with an enterokinase cleavage site immediately adjacent to thestart of the mature sequence, and purified exactly as for recombinanthuman Sonic hedgehog (See Example 9)) could be palmitoylated using theassay described in Example 2. Human Indian hedgehog was modified (SeeFIG. 1, lane h), indicating that palmitoylation is likely to be a commonfeature of hedgehog proteins. The ability to directly label Shh and Ihhwith radioactive palmitic acid in a cell-free system provided a simplescreen for amino acids involved in the modification reaction. Moreover,Indian hedgehog palmitoylated by the method described in Example 8 wassignificantly more potent in the C3H10T1/2 assay than the unmodifiedIhh.

EXAMPLE 8 Lipid Modifications of Sonic Hedgehog Using Acyl-Coenzyme A

The in vitro acylation of a protein containing an N-terminal cysteinecan be accomplished via a two-step, chemical reaction with a fattyacid-thioester donor. In the first step, the acyl group of the thioesterdonor transfers to the sulfhydryl of the N-terminal cysteine on theprotein by a spontaneous transesterification reaction. Subsequently, theacyl moiety undergoes a S to N shift to the α-amine of the N-terminalcysteine to form a stable amide bond. Direct acylation of an aminefunction on a protein may also occur with prolonged incubation with athioester, but the presence of a cysteine on the protein will acceleratethe reaction and allow control over the acylation site. In the presentexamples, commercially available Coenzyme A derivatives (Sigma ChemicalCompany, St. Louis Mo.) are utilized, but other thioester groups wouldalso achieve the same result. In fact, certain thioester leaving groups,such as thiobenzyl esters, would be expected to react more rapidly.Internal cysteine residues may also promote acylation to neighboringlysines (i.e., as in an internal cysteine-lysine pair) and this can beconveniently tested using synthetic peptides. Secondary acylationsoccuring on a protein during reaction with thioesters may be preventedby controlling the buffer composition, pH, or by site-directedmutagenesis of the neighboring lysines.

In preliminary analysis of the effect of acylation on the ability ofhuman Sonic hedgehog to induce alkaline phosphatase in C3H10T1/2 cells,reaction mixtures contained 1 mg/mL human Sonic hedgehog (51 μM), 500 μMof the particular, commercially available, acyl-Coenzyme A (compoundstested included acetyl-CoA (C2:0), butyryl-CoA (C4:0), hexanoyl-CoA(C6:0), octanoyl-CoA (C8:0), decanoyl-CoA (C10:0), lauroyl-CoA (C12:0),myristoyl-CoA (C14:0), palmitoyl-CoA (C16:0), palmitoleoyl-CoA (C16:1),stearoyl-CoA (C18:0), arachidoyl-CoA (C20:0), behenoyl-CoA (C22:0),lignoceroyl-CoA (C24:0), succinyl-CoA, and benzoyl-CoA), 25 mM DTT, and50 mM Na₂HPO₄ pH 7.0. The reactions were incubated at room temperaturefor 3 h and then analyzed immediately (without purification) forbioactivity in the C3H10T1/2 assay as described in Example 3. Samplesfor analysis by reverse phase HPLC and other physical methods wereusually stored at −70° C. HPLC analysis was carried out on a Vydac C₄reverse phase column (4.6 mm internal diameter×250 mm, 5 micronparticle) with a 40 min gradient of 5% acetonitrile to 85% acetonitrilein aqueous 0.1% TFA, at a flow rate of 1 mL/min. The effluent wasmonitored at 280 nm, and fractions were collected in some experimentsand analyzed for hedgehog protein on SDS-PAGE with detection byCoomassie staining and by Western blotting.

Comparison of the activity of the various reaction mixtures (FIG. 10)indicates that a chain length of between 12 and 18 carbons is optimal ininducing high alkaline phosphatase activity as compared to theunmodified protein. Increasing the chain length further resulted in anapparent reduction in activity, and the presence of a double bond in theunsaturated palmitoleoyl-CoA (C16:1) gave the same activity as the fullysaturated palmitoyl-CoA (C16:0). Upon reverse phase HPLC analysis of thereaction mixtures, we observed that many of the shorter chain lengthacyl-CoA derivatives had not reacted with the hedgehog protein, andtherefore the dependence of biological activity shown in FIG. 10 was nota true reflection of the acyl chain length.

In order to obtain data on the true activity of the modified proteins,and on the dependence of activity on acyl chain length, we developedmethods for the synthesis and purification of the individual N-terminalacylated forms. Palmitoylated, myristoylated, lauroylated, decanoylated,and octanoylated human Sonic hedgehog proteins, carrying a single acylchain attached to the α-amine of the N-terminal cysteine, were producedin reaction mixtures containing 0.80 mg/mL (41 μM) human Sonic hedgehog,410 μM (10-fold Molar excess) of either palmitoyl-CoA, myristoyl-CoA, orlauroyl-CoA, or 4.1 mM (100-fold Molar excess) of either decanoyl-CoA oroctanoyl-CoA, 25 mM DTT (for reaction mixtures containing palmitoyl-CoA,myristoyl-CoA, or lauroyl-CoA) or 0.5 mM DTT (for reaction mixturescontaining decanoyl-CoA or octanoyl-CoA), and 40 mM Na₂HPO₄ pH 7.0.Reaction mixtures were incubated at 28° C. for 24 h. Reaction of theN-terminal cysteine with the acyl thioesters results in the transfer ofthe acyl group to the sulfhydryl by a spontaneous transesterificationreaction, which is followed by a S to N shift to the α-amine to form astable amide linkage. The free sulflhydryl then undergoes a secondtransesterification reaction, yielding a protein with a fatty acyl groupattached via a thioester linkage to the sulfhydryl. The thioester-linkedacyl group was removed by adding consecutive 0.11 volume of 1 M Na₂HPO₄pH 9.0, and 0.11 volume of 1 M hydroxylamine (0.1 M final concentration)followed by incubation at 28° C. for 18 h, which leaves only the acylamide attached to the protein (62). 0.25 volume of 5% octylglucoside wasthen added (1% final concentration) and the mixture incubated for 1 h atroom temperature. The proteins were then purified in the presence of 1%octylglucoside using SP-Sepharose Fast Flow (Pharmacia) and Bio Scale S(Biorad) cationic ion exchange chromatographies. The purified proteinswere dialyzed against 5 mM Na₂HPO₄ pH 5.5, 150 mM NaCl, 1%octylglucoside, 0.5 mM DTT, and were stored at −70° C. The presence ofoctylglucoside was required to maintain full solubility; removal of thedetergent by dilution and dialysis resulted in a 75%, 41%, and 15% lossof the palmitoylated, myristoylated, and lauroylated proteins,respectively. ESI-MS of the HPLC-purified proteins confirmed theirintegrity: palmitoylated Sonic hedgehog, measured mass=19798, calculatedmass=19798.43; myristoylated Sonic hedgehog, measured mass=19770,calculated mass=19770.33; lauroylated Sonic hedgehog, measuredmass=19742, calculated mass=19742.33; decanoylated Sonic hedgehog,measured mass=19715, calculated mass=19714.28; octanoylated Sonichedgehog, measured mass=19686, calculated mass=19686.23.

Analysis of the various acylated forms of human Sonic hedgehog in theC3H10T1/2 assay (FIG. 11) indicated that the activity of the proteinswas dependent upon the chain length. The palmitoylated, myristoylated,and lauroylated proteins showed approximately equal activity with EC₅₀values of 5-10 ng/mL (100-200-fold increase in potency as compared tothe unmodified protein). Decanoylated human Sonic hedgehog, with an EC₅₀value of 60-70 ng/mL (15-30-fold increase in potency as compared to theunmodified protein), was less active than the palmitoylated,myristoylated, and lauroylated forms, while the octanoylated form wasthe least active with an EC₅₀ of 100-200 ng/mL (10-fold increase inpotency as compared to the unmodified protein). All of the acylatedforms were more potent than the unmodified protein which had an EC₅₀ of1000-2000 ng/mL. In addition to the decrease in EC₅₀, the palmitoylated,myristoylated, and lauroylated proteins induced approximately 2-foldmore alkaline phosphatase activity than the unmodified protein, whilethe decanoylated and octanoylated proteins induced approximately1.5-fold more.

In addition to the increase in potency of the myristoylated form ofhuman Sonic hedgehog observed in the C3H10T1/2 assay, this form issignificantly more potent than the unmodified protein at inducingventral forebrain neurons in explants of embryonic stage E11 rat braintelencephalon. Incubation of E11 telencephalic explants with variousconcentrations of unmodified, or myristoylated Sonic hedgehog, andsubsequent staining of the explants for the products of the dlx andislet-1/2 genes (markers of ventral forebrain neurons), indicates thatwhile induction by the unmodified protein is observed first at 48 nM,induction by the myristyolated form is observed first at 3 nM. Moreover,while the unmodified protein induces restricted expression at 3070 nM,the myristoylated protein induces widespread expression at only 48 nM. Asimilar increase in potency was observed when explants of embryonicstage E9 presumptive telencephalon were incubated with either theunmodified, or myristoylated proteins. Staining of the explants for theproduct of the Nkx2.1 gene (an early marker of ventral forebrainneurons), indicated that the unmodified protein induced Nkx2.1 first at384 nM, while for the myristoylated protein expression of Nkx2.1 wasobserved first at 12 nM. Moreover, at 48 nM myristoylated Sonichedgehog, expression of Nkx2.1 was widespread while it was undetectableat this concentration using the unmodified form.

Additionally, myristoylated human Sonic hedgehog has been shown to besignificantly more protective than the unmodified protein in reducingthe lesion volume which results from administration of malonate into thestriatum of the rat brain (See Example 16).

EXAMPLE 9 Chemical Derivatives of the N-terminal Cysteine of Human SonicHedgehog

A. General Methods

Alkylation of Proteins. Samples containing about 20 μg of the protein in50 μL of 6 M guanidine hydrochloride, 50 mM Na₂HPO₄ pH 7.0, were treatedwith 0.5 μL of 4-vinylpyridine for 2 h at room temperature. TheS-pyridylethylated protein was precipitated by addition of 40 volumes ofcooled ethanol. The solution was stored at −20° C. for 1 h and thencentrifuged at 14,000×g for 8 min at 4° C. The supernatants werediscarded and the precipitate was washed with cooled ethanol. Theprotein was stored at −20° C.

Peptide Mapping. Alkylated protein (0.4 mg/mL in 1 M guanidinehydrochloride, 20 mM Na₂HPO₄ pH 6.0) was digested with endo Lys-C (WakoPure Chemical Industries, Ltd.) at a 1:20 ratio. The digestion wasconducted at room temperature for 30 h. The reaction was stopped byacidification with 5 μL of 25% trifluoroacetic acid. The digest wasanalyzed on a Waters 2690 Separation Module with a Model 996 photodiodearray detector. Prior to injection, solid guanidine hydrochloride wasadded into the digest to a concentration of 6 M to dissolve insolublematerial. A reverse phase Vydac C₁₈ (2.1 mm internal diameter×250 mm)column was used for separation, with a 90 min gradient of 0.1%trifluoroacetic acid/acetonitrile and 0.1% trifluoroaceticacid/acetonitrile at a flow rate of 0.2 mL/min. Individual peaks werecollected manually for mass analysis.

Mass Determination. The molecular masses of intact proteins weredetermined by electrospray ionization mass spectroscopy (ESI-MS) on aMicromass Quattro II triple quadrupole mass spectrometer. Samples weredesalted using an on-line Michrom Ultrafast Microprotein Analyzer systemwith a Reliasil C₄ (1 mm internal diameter×50 mm) column. The flow ratewas 20 μL/min. All electrospray mass spectral data were processed usingthe Micromass MassLynx data system. The molecular masses of peptideswere determined by matrix assisted laser desorption ionizationtime-of-fight mass spectrometry (MALDI-TOF-MS) on a Voyager-DE™ STR(PerSeptive Biosystems, Framingham, Mass.). Sequencing of the modifedpeptide was performed by Post-source decay (PSD) measurement on the sameinstrument. α-Cyano-4-hydroxycinnamic acid was used as the matrix.

N-terminal Sequencing. Proteins were sequenced by Edman degradation on aPerkin-Elmer Applied Biosystems model 477A Pulsed-Liquid ProteinSequencer. PTH-thiaproline was made on line by directly loadingthiaproline (thiazolidine-4-carboxylic acid) into the sample loadingcartridge of the sequencer.

Bacterial expression and purification of wild type soluble human Sonichedgehog N-terminal fragment used for chemical modification. Bacterialpellets from cells expressing Shh at 4-5% of the total protein werethawed, resuspended in lysis buffer (25 mM Na₂HPO₄ pH 8, 150 mM NaCl, 1mM EDTA, 1 mM PMSF, 0.5 mM DTT) at a ratio of 1:4 (w/v) and lysed by twopasses through a Gaulin press (mfg. by APV Rannie, Copenhagen, Denmark)at 12,000 p.s.i. All subsequent purification steps were performed at2-8° C. unless indicated otherwise. The homogenate was centrifuged at19,000×g for 60 min and MES 0.5 M pH 5, was added to the resultinglysate at a ratio of 1:10 (v/v). The lysate (at pH 5.5) was loaded ontoan SP Sepharose Fast Flow (Pharmacia, Piscataway, N.J.) column (4 g E.coli wet weight/mL resin) equilibrated with 25 mM Na₂HPO₄ pH 5.5, 150 mMNaCl. The column was washed with 4 column volumes (CV) of equilibrationbuffer, then with 3 CV of 25 mM Na₂HPO₄pH 5.5, 200 mM NaCl, 0.5 mM DTT,and Histag-Shh was eluted with 800 mM NaCl in the same buffer. Elutionfractions were analyzed for absorbance at 280 nm and by SDS-PAGE.Imidazole (1 M stock solution at pH 7) and NaCl (5 M stock solution)were added to a pool of the peak Shh containing fractions from the SPSepharose eluate to give final concentrations of 20 mM and 1 Mrespectively, and this material was loaded onto a NTA-Ni agarose(Qiagen, Santa Clara, Calif.) column (20 mg/mL resin) equilibrated with25 mM Na₂HPO₄ pH 8, 1 M NaCl, 20 mM imidazole, 0.5 mM DTT. The columnwas washed with 5 CV of the same buffer and Histag-Shh eluted with 3 CV25 mM Na₂HPO₄pH 8, 1 M NaCl, 200 mM imidazole, 0.5 mM DTT. The proteincontent in the eluate pool from the NTA-Ni column was determined byabsorbance at 280 nm. The pool was warmed to room temperature and anequal volume of 2.5 M sodium sulfate was added. The Phenyl Sepharosestep was performed at room temperature. The material was loaded onto aPhenyl Sepharose Fast Flow (Pharmacia, Piscataway, N.J.) column (20mg/mL resin) equilibrated in 25 mM Na₂HPO₄ pH 8, 400 mM NaCl, 1.25 Msodium sulfate, 0.5 mM DTT. Histag-Shh was eluted with 25 mM Na₂HPO₄ pH8, 400 mM NaCl, 0.5 mM DTT. Typically, we recovered 2-3 g of His-taggedShh from 0.5 kg of bacterial paste (wet weight). The product wasfiltered through 0.2 μm filter, aliquoted, and stored at −70° C. TheHis-tagged Shh was about 95% pure as determined by SDS-PAGE. As afurther assessment of the characteristics of the purified product,samples were subjected to evaluation by electrospray ionization massspectrometry (ESI-MS). Approximately 50% of the protein was missing theN-terminal methionine.

To cleave off the hexahistidine tag, enterokinase (Biozyme, San Diego,Calif.) was incubated with the Histag-Shh at an enzyme:Shh ratio of1:1000 (w/w) for 2 h at 28° C. Uncleaved Histag-Shh and free Histag wereremoved by passing the digest through a second NTA-Ni agarose column (20mg Shh/niL resin). Prior to loading, imidazole (1 M stock solution at pH7) and NaCl (5M stock solution) were added to the digest to give finalconcentrations of 20 mM and 600 mM, respectively. This material wasloaded onto a NTA-Ni column equilibrated in 25 mM Na₂HPO₄ pH 8, 600 mMNaCl, 20 mM imidazole, 0.5 mM DTT and the flow through collected. Thecolumn was washed with 1 CV of the same buffer and pooled with the flowthrough. MES (0.5 M stock solution at pH 5) was added to the NTA-Niagarose unbound fraction to a final concentration of 50 mM and twovolumes of water were added. This material was loaded onto a second SPSepharose Fast Flow column (20 mg/mL resin) equilibrated with 5 mMNa₂HPO₄pH 5.5, 150 mM NaCl, 0.5 mM DTT. The column was washed with 3 CVof equilibration buffer and 1 CV of the same buffer containing 300 mMNaCl. Shh was eluted with 5 mM Na₂HPO₄ pH 5.5, 800 mM NaCl, 0.5 mM DTT.Atomic absorption data revealed that Shh at this stage contained 0.5 molequivalent of Zn²⁺. An equimolar concentration of ZnCl₂ was added to theShh eluant and the protein dialyzed against 5 mM Na₂HPO₄pH 5.5, 150 mMNaCl, 0.5 mM DTT. The resulting Shh was >98% pure as characterized bySDS-PAGE, size exclusion chromatography (SEC), and ESI-MS and, by atomicabsorption, contained between 0.9 and 1.1 Zn²⁺/Shh.

ESI-MS data for Histag Shh and products resulting after removal of thehistag are summarized in Table 7. TABLE 7 Characterization of Shh byESI-MS. Mass (Da) Protein Calculated Measured Histag-Shh (-Met) 21433.8221434 (Intact) 21565.01 21565 Enterokinase-cleaved Shh 19560.02 19560B. Specific Chemical Modifications

Modification of human Sonic hedgehog with N-ethylmaleimide. Purified Shhin 5 mM Na₂HPO₄ pH 5.5, 150 mM NaCl, 0.5 mM DTT was treated with 10 mMN-ethylmaleimide for 1 h on ice and then dialyzed into 5 mM Na₂HPO₄ pH5.5, 150 mM NaCl. The MALDI-TOF-MS data showed that the N-ethylmaleimide(NEM)-modified protein had an increase in mass of 126 Da, whichindicates that only one cysteine residue in Shh was modified by thereagent. N-terminal sequencing data showed that the protein issequencible and that an unusual peak, probably PTH-NEM-Cys related, wasdetected at the first cycle (data not shown). Mass spectrometricanalysis of the pyridylethylated-NEM-Shh under denaturing conditionsshowed that only two cysteine residues in the protein were alkylated,confirming that only the thiol group of the N-terminal cysteine residuewas modified by NEM under native conditions (Table 8). The other twocysteine residues, which are apparently buried in the hydrophobic coreof the protein, cannot be modified without prior denaturation. TABLE 8Characterization of NEM-modified Shh by MS. Mass Mass Protein(Calculated) (Measured) Pyridylethylated NEM Shh 19895 Da if containing2 free Cys residues 19895 Da if containing 3 free Cys residues 20000 Da

When tested in the C3H10T1/2 assay (See Example 3) theN-ethylmaleimide-modified hedgehog protein was equal in activity to theunmodified protein. This demonstrates that a free sulfhydryl at theN-terminus of hedgehog is not required for activity and that theN-ethylmaleimide moiety is hydrophobic enough to confer some activity onhedgehog compared to other more hydrophilic modifications, such asconversion of Cys-1 to His or Asp, which produce a reduction inactivity. Modification of human Sonic hedgehog with formaldehyde to forman N-terminal thiaproline, and with acetaldehyde and butyraldehyde toform N-terminal thiaproline derivatives. For formaldehyde modification,purified Shh at 3 mg/mL in 5 mM Na₂HPO₄ pH 5.5, 150 mM NaCl, 0.5 mM DTTwas treated with 0.1% formaldehyde, with or without 10% methanol, atroom temperature for 1 to 6 h. The protein was either dialyzed against 5mM Na₂HPO₄ pH 5.5, 150 mM NaCl, or was purified on a CM-Poros column(Perseptive Biosystems) as described below and then dilayzed against 5mM Na₂HPO₄ pH 5.5, 150 mM NaCl. For modification with acetaldehyde orbutyraldehyde, purified Shh at 3 mg/mL in 5 mM Na₂HPO₄ pH 5.5, 150 mMNaCl, 0.5 mM DTT was treated with 0.1% acetaldehyde or butyraldehyde atroom temperature for 1 h and then the protein purified on a CM-Poroscolumn. ESI-MS data for the formaldehyde, acetaldehyde-, andbutyraldehyde-treated forms of the protein indicated that their masseswere 13 Da, 27 Da, and 54 Da higher, respectively, than the unmodifiedprotein (Table 9). TABLE 9 Expected and observed masses of human Sonichedgehog treated with formaldehyde, acetaldehyde, and butyraldehyde.Expected mass* Observed mass* Protein (MH⁺) (MH⁺) Unmodified 19560.0219560 Formaldehyde-treated 19572.03 19573 Acetaldehyde-treated 19586.0619587 Butyraldehyde-treated 19614.11 19614*Average masses were used in calculating the expected masses

For the formaldehyde-treated protein, peptide mapping, as describedabove, demonstrated that the site of the modification occurred in thepeptide spanning the first 9 N-terminal residues, and that the exactmass increase was 12 Da. The results of MALDI-PSD MS studies of thispeptide indicated that the modification occurred on Cys-1, and could beexplained by a modification of the N-terminal o-amine and the thiol sidechain of Cys-1 to form a thiaproline (See FIG. 12). The structure of thethiaproline was confirmed by automated N-terminal Edman sequencing using“on-line” prepared PTH-thiaproline as a standard. For the acetaldehyde-and butyraldehyde-treated proteins, the ESI-MS data were consistent withthe modifications occuring by means of the same chemistry as for thereaction with formaldehyde, although the exact site of modification hasnot been established. When tested in the C3H10T1/2 cell-based assay, theformaldehyde-, acetaldehyde-, and butyraldehyde-modified proteins wereapproximately 8-fold, 2-fold, and 3-fold, respectively, more potent thanunmodified Shh.

Modification of human Sonic hedgehog with N-isopropyliodoacetamide. Thisexample shows that modification of human Shh with a hydrophobicderivative of iodoacetamide can enhance the potency of the protein ascompared to the unmodified Shh. Purified Shh (1 mg/mL in 5 mM Na₂HPO₄ pH7.0, 150 mM NaCl, 0.1 mM DTT) was incubated with 1 mMN-isopropyliodoacetamide (NIPIA) at 4° C. for 18 h. DTT was then addedto 10 mM final concentration and the sample was dialyzed extensivelyagainst 5 mM Na₂HPO₄ pH 5.5, 150 mM NaCl, 0.5 mM DTT. The sample waspurified on SP Sepharose Fast Flow resin and dialyzed further against 5mM Na₂HPO₄ pH 5.5, 150 mM NaCl, 0.5 mM DTT. ESI-MS data indicatedcomplete conversion to a species with a mass of 19660, corresponding tothe predicted mass value (19659) for the singly modified protein.Specific modification of the N-terminal cysteine was confirmed bypeptide mapping of proteolytic fragments. When tested in the C3H10T1/2cell-based assay, the NIPIA-modified human Shh was approximately 2-foldmore potent than the unmodified protein. While the modification of theprotein resulted in only a modest increase in potency, it is expectedthat modification of the protein with long chain alkyl iodoacetamidederivatives will result in hydrophobically-modified forms of the proteinwith much greater increases in potency, possibly akin to the100-200-fold increase observed for the palmitoylated, myristoylated, andlauroylated Shh proteins (See Example 8).

Modification of human Sonic hedgehog with 1-bromo-2-butanone to form asix-membered hydrophobic ring at the N-terminus. Athiomorpholinyl-(tetrahydrothiazinyl-)derivative of Shh was prepared byincubating human Shh-N (3 mg/mL in 5 mM Na₂HPO₄ pH 5.5, 150 mM NaCl,0.15 mM DTT) with 11 mM 1-bromo-2-butanone at room temperature for 60min, followed by reduction with 5 mM NaCNBH₃ at room temperature for 60min. The reaction product was purified on a CM-Poros column (PerseptiveBiosystems) as described below and was dialyzed against 5 mM Na₂HPO₄ pH5.5, 150 mM NaCl, 0.5 mM DTT. ESI-MS and proteolytic peptide mappingdata indicated that the product was a mixture of the expectedthiomorpholinyl derivative (calculated mass=19615, observed mass=19615)and two forms of the protein both with 16 additional mass units. One ofthese forms is presumably the uncyclized keto-thioether intermediate.The mixture was tested in the C3H10T1/2 assay which indicated that itwas approximately 5-fold more potent than the unmodified protein.

EXAMPLE 10 Genetically Engineered Mutations of Human Sonic Hedgehog

A. Genetically Engineered Mutations of the N-terminal Cysteine

In this example, we show that specific replacement of the N-terminalcysteine of human Sonic hedgehog (Cys-1) by single and multiplehydrophobic amino acid residues results in increased potency as comparedto the wild type protein in the C3H10T1/2 cell-based assay described inExample 3.

Construction of Shh Cys-1 mutants. The 584 bp NcoI-XhoI restrictionfragment carrying the His-tagged wild type Shh N-terminal fragment fromp6H-SHH was subcloned into the pUC-derived cloning vector pNNO5 toconstruct the plasmid pEAG649. Cys-1 mutants of soluble human Shh weremade by unique site elimination mutagenesis of the pEAG649 plasmidtemplate using a Pharmacia kit following the manufacturer's recommendedprotocol. In designing the mutagenic primers, if a desired mutation didnot produce a restriction site change, a silent mutation producing arestriction site change was introduced into an adjacent codon tofacilitate identification of mutant clones following mutagenesis. Toavoid aberrant codon usage, substituted codons were selected from thoseoccurring at least once elsewhere in the human Shh cDNA sequence. Thefollowing mutagenic primers were used: (1) for C1F: 5′ GGC GAT GAC GATGAC AAA TTC GGA CCG GGC AGG GGG TTC 3′ (SEQ ID NO:______), whichintroduces an Apol site to make pEAG837; (2) for C1I: 5′ GGC GAT GAC GATGAC AAA ATA GGA CCG GGC AGG GGG TTC 3′ (SEQ ID NO:______), which losesan RsrII site to make pEAG838; and (3) for C1M: 5′ GGC GAT GAC GAT GACAAA ATG GGC CCG GGC AGG GGG TTC GGG 3′ (SEQ ID NO:______), which losesboth RsrII and AvaII sites to make pEAG839. Mutations were confirmed byDNA sequencing through a 180 bp NcoI-BgIII restriction fragment carryingthe mutant SHH proteins' N-termini in plasmids pEAG837-839. Expressionvectors were constructed by subcloning each mutant plasmid's 180 bpNcoI-BgIII fragment and the 404 bp BgIII-XhoI fragment from pEAG649 intothe phosphatase-treated 5.64 kb XhoI-NcoI pET11d vector backbone ofp6H-SHH. Presence of the introduced restriction site change wasreconfirmed in the expression vector for each Cys-1 mutant (C1F inpEAG840, C1I in pEAG841, and C1M in pEAG842). Expression vectors weretransformed into competent E. coli BL21(DE3)pLysS (Stratagene) followingthe manufacturer's recommended protocol and selected on LB agar platescontaining 100 μg/mL ampicillin and 30 μg/mL chloramphenicol. Individualcolonies were selected and transformed bacteria were grown to an A₆₀₀ of0.4-0.6 and induced for 3 h with 0.5 mM IPTG. Bacterial pellets wereanalyzed for expression of the mutant proteins by reducing SDS-PAGE andby Western blotting.

A soluble human Shh mutant with multiple N-terminal hydrophobicsubstitutions (C1II) was made by unique site elimination mutagenesisusing a Pharmacia kit following the manufacturer's recommended protocol.In designing the mutagenic primers, if a desired mutation did notproduce a restriction site change, a silent mutation producing arestriction site change was introduced into an adjacent codon tofacilitate identification of mutant clones following mutagenesis. Toavoid aberrant codon usage, substituted codons were selected from thoseoccurring at least once elsewhere in the human Shh cDNA sequence. Thefollowing mutagenic primer was used on the C1F template plasmid pEAG837for C1II: 5′ GCG GCG ATG ACG ATG ACA AAA TCA TCG GAC CGG GCA GGG GGT TCGGG 3′ (SEQ ID NO:______), which removes an Apol site to make pEAG872.Mutations were confirmed by DNA sequencing through a 0.59 kb NcoI-XhoIrestriction fragment carrying the mutant C1II Shh. An expression vectorwas constructed by subcloning the mutant plasmid's NcoI-XhoI fragmentinto the phosphatase-treated 5.64 kb XhoI-NcoI pET11d vector backbone ofp6H-SHH. Presence of the introduced restriction site change wasreconfirmed in the expression vector for the C1I mutant, pEAG875. Theexpression vector was transformed into competent E. coli BL21(DE3)pLysS(Stratagene) following the manufacturer's recommended protocol andselected on LB agar plates containing 100 μg/mL ampicillin and 30 μg/mLchloramphenicol. Individual colonies were selected and transformedbacteria were grown to an A₆₀₀ of 0.4-0.6 and induced for 3 h with 0.5mM IPTG. Bacterial pellets were analyzed as described above to confirmexpression of mutant Shh protein.

Purification of Cys-1 mutants of human Sonic hedgehog. The His-taggedmutant hedgehog proteins were purified from the bacterial pellets asdescribed for the wild type protein above except for two modifications.(1) The Phenyl Sepharose step was eliminated and instead the proteinpool from the first NTA-Ni agarose column was dialyzed into 25 mMNa₂HPO₄ pH 8, 400 mM NaCl, 0.5 mM DTT in preparation for theenterokinase cleavage step. (2) The final ion exchange step was changedfrom step elution on SP-Sepharose Fast Flow to gradient elution from aCM-Poros column (Perseptive Biosystems). This was carried out in 50 mMNa₂HPO₄ pH 6.0 with a 0-800 mM NaCl gradient over 30 column volumes. Thepooled peak fractions from this step were dialyzed into 5 mM Na₂HPO₄ pH5.5, 150 mM NaCl and were stored at −80° C. Mass spectrometry of thepurified proteins gave the predicted mass ions for each purified form.

Activity of the Cvs-1 mutants of human Sonic hedgehog. As shown in Table10, mutation of the N-terminal cysteine has a significant effect on thepotency of the resulting hedgehog protein in the C3H10T1/2 assay. Forsingle changes, potency generally correlates with the hydrophobicity ofthe substituted amino acid, that is phenylalanine and isoleucine givethe greatest activation, methionine is less activating, while histidineand aspartic acid diminish activity compared to the wild type cysteine.Replacing the cysteine with two isoleucines gives an additional increasein activity over the single isoleucine substitution. Given that nineamino acids are categorized as more hydrophobic than cysteine (Proteins:structures and molecular properties, 2^(nd) ed, 1993, T. E. Creighton,W. H. Freeman Co. page 154), the substitutions tested above are clearlynot an exhaustive survey of the possible mutations at the N-terminusthat can give rise to more active forms of hedgehog. However, theresults demonstrate that activation is not restricted to a single aminoacid structure and that substitution of more than one amino acid cangive a further increase in potency. Therefore, one skilled in the artcould create forms of hedgehog with other amino acid substitutions atthe N-terminus that would be expected to have greater potency than thewild type protein. TABLE 10 Relative potency of amino acid modificationsof human Sonic hedgehog in the C3H10T1/2 assay. N-TERMINUS RELATIVEPOTENCY C (wild type) 1× M 2× F 4× I 4× II 10× B. Genetically Engineered Mutations of Internal Residues

Construction of the C1II/A169C mutant. The soluble human Shh mutantC1/A169C (with cysteine substituted for the dispensable C-terminalresidue A169 which is predicted to have a high fractional solventaccessibility) was made by unique site elimination mutagenesis using aPharmacia kit following the manufacturer's recommended protocol andemploying the mutagenic oligo design principles described above. Thefollowing mutagenic primer 5′ GAG TCA TCA GCC TCC CGA TTT TGC GCA CACCGA GTT CTC TGC TTT CAC C 3′ (SEQ IDNO:______) was used on C1II Shhtemplate pEAG872 to add an FspI site to make pSYS049. The C1II/A169Cmutations were confirmed by DNA sequencing through a 0.59 kb NcoI-XhoIrestriction fragment. The expression vector pSYS050 was constructed bysubcloning the NcoI-XhoI fragment into the phosphatase-treated 5.64 kbXhoI-NcoI pET11d vector backbone of p6H-SHH. Presence of the introducedrestriction site change was reconfirmed in the expression vector. Theexpression vector was transformed into competent E. coli BL21(DE3)pLysS,colonies were selected, induced, and screened for Shh expression asdescribed above.

Purification of the C1II/A169C mutant. The C1II/A169C mutant waspurified as described in Example 9 for wild type Shh except with thefollowing modifications. (1) EDTA was left out of the lysis buffer, (2)the order of the NTA-Ni and SP Sepharose steps were switched and thePhenyl Sepharose step was omitted, (3) after clarification of the lysedbacteria by centrifugation, additional NaCl was added to the supernatantto a final concentration of 300 mM, (4) the elution buffer from theNTA-Ni column contained 25 mM Na₂HPO₄ pH 8.0, 200 mM imidazole, 400 mMNaCl, (5) the elution pool from the NTA-Ni column was diluted with 3volumes of 100 mM MES pH 5.0 prior to loading onto the SP Sepharosecolumn, (6) prior to addition of enterokinase, the SP Sepharose elutionpool was diluted with half a volume of 50 mM Na₂HPO₄ pH 8.0, and (7) theDTT in the elution buffer from the final SP Sepharose column contained0.2 mM DTT and the elution pool from this step was aliquoted and storedat −70° C.

Hydrophobic modification and activity of the C1II/A169C mutant. Formodification with N-(1-pyrene) maleimide (Sigma), purified C1II/A169C at4.6 mg/mL in 5 mM Na₂HPO₄ pH 5.5, 800 mM NaCl, 0.2 mM DTT was dilutedwith an equal volume of 50 mM MES pH 6.5 and to this a twentieth of avolume of pyrene maleimide from a 2.5 mg/mL stock in DMSO was added. Thesample was incubated for 1 h at room temperature in the dark. At thistime additional DTT was added to 0.5 mM and the sample incubated furtherfor an additional hour at room temperature. The modified protein wastested directly for activity in the C3H10T1/2 assay as described inExample 3. Prior to modification, the specific activity of the proteinwas EC₅₀=0.22 μg/mL, while after treatment with pyrene maleimide thespecific activity was increased to EC₅₀=0.08 μg/mL. Increases in thespecific activity of the modified product by up to 3-fold were observedfrequently indicating that the addition of the hydrophobic group nearthe C-terminus of Shh resulted in a further increase in activity ascompared to the C1II starting material. When compared to the wild typeunmodified Sonic hedgehog protein, the N-(1-pyrene) maleimide-modifiedC1II protein was approximately 30-fold more potent. While pyrenemaleimide provided a simple test system for evaluating modification atthis site, other hydrophic maleimides or other cysteine targetedchemistries can also be used.

EXAMPLE 11 Comparison of the Potency of Various Hydrophobically-ModifiedForms of Human Sonic Hedgehog in the C3H10T1/2 Assay

The activity of various hydrophobically-modified forms of human Sonichedgehog (prepared using the chemistries and genetic engineeeringmethods described in Section V) was tested in the C3H10T1/2 assay asdescribed in Example 3.

The derivatives were assayed over a concentration range as described inExample 3. The concentration of hedgehog derivative that resulted in 50%of the maximum response in the assay was compared to the wild typeconcentration. The relative activities are shown in Table 11, below, andin FIG. 13. TABLE 11 Relative Potency of Hedgehog Derivatives in theC3H10T1/2 assay. EC₅₀ (x-fold more potent Modification than wild typeShh) C:16 palmitoyl 100 C:14 myristoyl 100 C:12 lauroyl 100 C:10decanoyl 33 Isoleucyl-isoleucyl with A169C pyrenyl 30 C:8 octanoyl 10Isoleucyl-isoleucyl 10 C:0 thiaprolyl 8 Thiomorpholinyl 5 Phenylalanyl 4Isoleucyl 4 N-isopropylacetamidyl 2 Methionyl 2 N-ethylmaleimidyl 1Cysteinyl (wild type) 1 Aspartyl <1 Histidyl <1

The C3H10T1/2 assay demonstrates that a wide variety of hydrophobicmodifications to hedgehog increase the protein's activity when comparedto the wild type, unmodified protein. Hydrophilic modifications(aspartic acid and histidine) do not have this effect.

EXAMPLE 12 Evaluating the Efficacy of Hydrophobically-Modified HumanSonic Hedgehog in a Rat Malonate-Induced Striatal Lesion Assay

Injection of malonate, an inhibitor of the mitochondrial enzymesuccinate dehydrogenase, into the rat striatum (the rodent equivalent ofthe primate caudate and putamen) causes degeneration of striatal mediumspiny neurons. In humans, degeneration of medium spiny neurons in thecaudate and putamen is the primary pathological feature of Huntington'sdisease. Thus, the malonate-induced striatal lesion in rats can be usedas a model to test whether hydrophobically-modified hedgehog proteinscan prevent the death of the neurons which degenerate in Huntington'sdisease.

Sprague-Dawley rats were injected with various concentrations ofhydrophobically-modified human Sonic hedgehog in the striatum usingstereotaxic techniques. Stereotaxic injections (2 μL) were performedunder sodium pentobarbital anesthesia (40 mg/kg) and placed at thefollowing coordinates: 0.7 mm anterior to bregma, 2.8 mm lateral to themidline, and 5.5 mm ventral to the surface of the skull at bregma. Atvarious times (usually 48 h) after injection of thehydrophobically-modified protein, rats were anesthetized with isofluraneand given a stereotaxic injection of malonate (2 μmol in 2 μL) at thesame coordinates in the striatum. Four days after malonate injection,rats were sacrificed and their brains removed for histological analysis.Coronal sections were cut through the striatum at a thickness of 25 μmand stained for cytochrome oxidase activity to distinguish lesioned fromunlesioned tissue. The volume of the lesion in the striatum is measuredusing an image analysis system.

The effect of hydrophobically-modified human Sonic hedgehog protein inthe malonate-induced rat striatal lesion model is shown in FIG. 14.Unmodified Sonic hedgehog (prepared as described in Example 9),myristoylated Shh (prepared as described in Example 8), and the C1IImutant of Shh (prepared as described in Example 10) all reduced lesionvolume to a similar extent in this model. However, thehydrophobically-modified proteins (myristoylated Shh and C1II Shh)showed an increase in potency relative to the unmodified Sonic hedgehog.

EXAMPLE 13 N-Octylmaleimide Derivitization of sHh-N

For a 1 mg/ml Final Concentration

1) Make a 20 mM solution of octylmaleimide (m.w.=209) in DMSO (˜4.2mg/ml).

2) Dilute stock of 10 mg/ml sHh-N (in 5 mM NaPO4 pH 5.5, 150 mM NaCl,0.5 mM DTT) 10-fold with PBS (Gibco product #20012-027, pH 7.2) to givea 1 mg/ml (or 50 uM) sHh-N solution. [NOTE: DTT, which competes withsHh-N for maleimide in the subsequent reaction, is also 50 μM in thissolution.]

3) Immediately add 1/200 vol. of octylmaleimide to the 1 mg/ml sHh-N(i.e. 5 μl/1 ml). This gives a 2:1 molar ratio (100 μM:50 μM) ofoctylmaleimide to sHh-N.

4) Mix this solution by gentle inversion of the tube and incubate for 1hour at room temperature.

5) Finally, 1/1000 vol. of 0.35 M DTT was added to each tube to scavengeany remaining octylmaleimide and to serve as a reductant.

6) For a vehicle control, combine a solution of vehicle (5 mM NaPO4 pH5.5, 150 mM NaCl, 0.5 mM DTT) with PBS (Gibco product #20012-027, pH7.2) in a 1:10 ratio. Add 1/400 vol. of 20 mM octylmaleimide in DMSO anda 1/400 vol. of DMSO to give a final concentration of 50 μMN-octylmaleimide and 0.5% DMSO. Finally, add 1:1000 vol. of 0.35 M DTT.

Approximate Composition of the 1 mg/ml N-octylmaleimide sHh-N Solution

-   PBS (˜pH 7.2)-   50 μM sHh-N conjugated to N-octylmaleimide-   50 μM DTT conjugated to N-octylmaleimide-   350 μM DTT-   0.5% DMSO    Approximate Composition of the N-octylmaleimide Vehicle Solution-   PBS (˜pH 7.2)-   50 μM DTT conjugated to N-octylmaleimide-   350 μM DTT-   0.5% DMSO    For a 3 mg/ml Final Concentration

1) Make a 60 mM solution of octylmaleimide (m.w.=209) in DMSO (˜12.6mg/ml).

2) Dilute stock of 10 mg/ml sHh-N (in 5 mM NaPO4 pH 5.5, 150 mM NaCl,0.5 mM DTT) 10-fold with PBS (Gibco product #20012-027, pH 7.2) to givea 3 mg/ml (or 150 uM) sHh-N solution. [NOTE: DTT, which competes withsHh-N for maleimide in the subsequent reaction, is also 150 μM in thissolution.]

3) Immediately add 1/200 vol. of octylmaleimide to the 3 mg/ml sHh-N(i.e. 5 μl/1 ml). This gives a 2:1 molar ratio (300 μM:150 μM) ofoctylmaleimide to sHh-N.

4) Mix this solution by gentle inversion of the tube and incubate for 1hour at room temperature.

5) Finally, add 1/1000 vol. of 0.35 M DTT to each tube to scavenge anyremaining octylmaleimide and to serve as a reductant.

6) For a vehicle control, combine a solution of vehicle (5 mM NaPO4 pH5.5, 150 mM NaCl, 0.5 mM DTT) with PBS (Gibco product #20012-027, pH7.2) in a 3:7 ratio. Add 1/400 vol. of 60 mM octylmaleimide in DMSO anda 1/400 vol. of DMSO to give a final concentration of 150 μMN-octylmaleimide and 0.5% DMSO. Finally, add 1:1000 vol. of 0.5 M DTT.

Approximate Composition of the 3mg/ml N-octylmaleimide sHh-N Solution

-   PBS (˜pH 7.2)-   150 μM sHh-N conjugated to N-octylmaleimide-   150 μM DTT conjugated to N-octylmaleimide-   500 μM DTT-   0.5% DMSO    Approximate Composition of the N-octylmaleimide Vehicle Solution-   PBS (˜pH 7.2)-   150 μM DTT conjugated to N-octylmaleimide-   500 μM DTT-   0.5% DMSO-   0.5% DMSO    References-   1. Perrimon, N. (1995) Cell 80, 517-520-   2. Johnson, R. L., and Tabin, C. (1995) Cell 81, 313-316-   3. Riddle, R. D. et al. (1993) Cell 75, 1401-1416-   4. Niswander, L., et al. (1994) Nature 371, 609-612-   5. Laufter, E., et al. (1994) Cell 79. 993-1003-   6. Roberts, D. J., et al. (1995) Development 121, 3163-3174-   7. Chiang, C., et al. (1996) Nature 382, 407-413-   8. Bellusci, S., et al. (1997) Development 124, 53-63-   9. Marigo, V., et al. (1996) Nature 384, 176-179-   10. Stone, D. M., et al. (1996) Nature 384, 129-134-   11. Alcedo, J., et al. (1996) Cell 86, 221-232.-   12. Dominguez, M., et al. (1996) Science 272, 1621-1625-   13. Alexandre, C., et al. (1996) Genes & Dev. 10, 2003-2013-   14. Therond, P. P., et al. (1996) Proc. Natl. Acad. Sci. USA 93,    4224-4228-   15. Lee, J. J., et al. (1994) Science 266, 1528-1536-   16. Bumcrot, D. A., et al. (1995), Mol. Cell Biol. 15, 2294-2303-   17. Porter, J. A., et al. (1995) Nature 374, 363-366-   18. Porter, J. A., et al. (1996) Science 274, 255-258-   19. Porter, J. A., et al. (1995) Cell 86, 21-34-   20. Marigo, V., et al. (1995) Genomics 28, 44-51-   21. Sanicola, M., et al. (1997) Proc. Natl. Acad. Sci. USA 94,    6238-6243.-   22. Spengler, B., et al. (1992) Rapid. Commun. Mass Spectrom. 6,    105-108.-   23. Spengler, B., et al. (1992) J. Phys. Chem. 96, 9678-9684-   24. Caron, J. M. (1997) Mol. Biol. Cell 8, 621-636-   25. Kinto, N., et al. (1997) FEBS Letts. 404, 319-323-   26. Ericson, J., et al. (1996) Cell 87, 661-673-   27. Wen, D., et al. (1996) Biochemistry 30, 9700-9709-   28. Bizzozero, O. A. (1996) Meth. Enzymol. 250, 361-382-   29. Wedegaertner, P. B., et al. (1995) J. Biol. Chem. 270, 503-506-   30. Grosenbach, D. W., et al. (1997) J. Biol. Chem. 272, 1956-1964-   31. Pepinsky, R. B., et al. (1991) J. Biol. Chem. 266, 18244-18249-   32. Tanaka Hall, T. M., et al. (1995) Nature 378, 212-216-   33. Mohler and Vani, (1992) Development 115, 957-971-   34. Hall et al., (1995) Nature 378, 212-216-   35. Ekker et al., (1995) Current Biology 5, 944-955-   36. Fan et al., (1995) Cell 81, 457-465-   37. Chang et al., (1994) Development 120, 3339-3353-   38. Echelard et al., (1993) Cell 75, 1414-1430-   39. Ericson et al., (1995) Cell 81, 747-756-   40. Zoeller et al., (1984) Proc. Natl. Acad. Sci. USA, 81, 5662-66-   41. Kaufman and Sharp, (1982) Mol. Cell. Biol., 2, 1304-1319-   42. Leung et al., (1989) Technique 1, 11-15-   43. Mayers et al., (1989) Science 229, 242-   44. Harang, S. A., (1983) Tetrahedron 39, 3-   45. Itakura et al., (1984) Ann. Rev. Biochem. 53, 323-   46. Cunningham and Wells, (1989) Science 244, 1081-1085-   47. Adelman et al., (1983) DNA 2, 183-   48. Wells et al., (1985) Gene 34, 315-   49. W. D. Huse et al., (1989) Science 246, 1275-1281-   50. H. L. Yin and T. P. Stossel, (1979) Nature 281, 583-586-   51. Lindley, (1956) Nature 178, 647-   52. Gross and Witkip, (1961) J. Am. Chem. Soc. 83, 1510-   53. D. M. Haverstick and M. Glaser, (1987) Proc. Natl. Acad. Sci.    USA 64, 4475-4478-   54. Szoka et al., (1980) Ann. Rev. Biophys. Bioeng. 9, 467-508-   55. Ohsawa et al., (1984) Chem. Pharm. Bull. 32, 2442-   56. Batzri et al., (1973) Biochim. Biophys. Acta 298, 1015-1019-   57. Szoka et al., (1978) Proc. Natl. Acad. Sci. USA 75, 4191-4199-   58. Pick, (1981) Arch. Biochem. Biophys. 212, 186-194-   59. Kasahara et al., (1977) J. Biol. Chem. 251, 7384-7390-   60. Racker et al., (1979) Arch. Biochem. Biophys. 198, 470-477-   61. Papahadjopoulos et al., (1991) Proc. Natl. Acad. Sci. USA 88,    11460-11464-   62. Chou, T. C. and Lipmann, F. (1952) J. Biol. Chem. 196, 89-   63. Kaminski et al., (1993) New Engl. J Med. 329, 459-   64. Tomac et al., (1995) Nature 373, 335-339-   65. Gash et al., (1996) Nature 380, 252-255-   66. Hoffer et al., (1994) Neuroscience Lett. 182, 107-111-   67. Duchen, L. W. and Strich, S. J., (1968), J. Neurol. Neurosurg.    Psychiatry 31, 535-542-   68. Kennel et al., (1996) Neurobiology of Disease 3, 137-147-   69. Ripps et al., (1995) Proc. Natl. Acad. Sci, USA, 92: 689-693-   71. Nicholson, L. et al., (1995) Neuroscience 66, 507-521-   72. Beal, M. F. et al., (1993) J. Neuroscience 13, 4181-4192-   73. Davies, S. et al., (1997) Cell 90, 537-548-   74. Hebr-Katz, R. (1993) Int. Rev. Immunol. 9, 237-285-   75. Borg et al., (1990) Brain Res., 518, 295-298-   76. Apfel et al., (1991) Ann. Neurol., 29, 87-90-   77. Noren, C. J. et al., (1989) Science 244, 182-188-   78. Thorson, J. S. et al., (1998) Methods Mol. Biol. 77, 43-73-   79. Das, A. K. et al., (1997) J. Biol. Chem. 272, 11021-11025-   80. Berthiaume, L. & Resh, M. D. (1995) J. Biol. Chem. 270,    22399-22405-   81. Raju, R. V. et al., (1995) Mol. Cell. Biochem. 149-150, pp.    191-202-   82. Duronio, R. J. et al., (1993) in Lipid Modification of    Proteins, M. J. Schlesinger, ed.-   83. Krutsch, H. C. & Inman, J. K. (1993) Anal. Biochem. 209, 109-116-   84. Heitz, J. R. et al., (1968) Arch. Biochem. Biophys. 12, 627-636-   85. Stefanini, S. et al., (1972) Arch. Biochem. Biophys 151, 28-34-   86. Kawaguchi, A. J. (1981) Biochem. (Tokyo) 89, 337-339-   87. House, H. O. (1972) in Modem Synthetic Reactions, W. A.    Benjamin, ed.

All of the above-cited references and publications are herebyincorporated by reference.

Equivalents

While we have described a number of embodiments of this invention, it isapparent to persons having ordinary skill in the art that our basicembodiments may be altered to provide other embodiments that utilize thecompositions and processes of this invention. Therefore, it will beappreciated that the scope of this invention includes all alternativeembodiments and variations which are defined in the foregoingspecificafion and by the claims appended hereto; and the invention isnot to be limited by the specific embodiments presented in the examples.

1. An isolated, protein comprising an N-terminal amino acid and aC-terminal amino acid, wherein the protein is selected from the groupconsisting of: (a) a protein with an N-terminal cysteine that isappended with at least one hydrophobic moiety; (b) a protein with anN-terminal amino acid that is not a cysteine appended with at least onehydrophobic moiety; and (c) a protein with at least one hydrophobicmoiety substituted for the N-terminal amino acid.
 2. The protein ofclaim 1, wherein the hydrophobic moiety is a peptide comprising at leastone hydrophobic amino acid.
 3. The protein of claim 1, wherein thehydrophobic moiety is a lipid.
 4. The protein of claim 1, wherein theprotein further comprises a hydrophobic moiety substituted for, orappended to, the C-terminal amino acid.
 5. The protein of claim 1,wherein the protein is an extracellular signaling protein.
 6. Theprotein of claim 1, wherein the N-terminal amino acid is a functionalderivative of a cysteine.
 7. The protein of claim 1, wherein the proteinis modified at both the N-terminal amino acid and the C-terminal aminoacid.
 8. The protein of claims 4 or 7, wherein the protein has ahydrophobic moiety substituted for, or appended to, at least one aminoacid intermediate to the N-terminal and C-terminal amino acids.
 9. Theprotein of claim 1, wherein the protein has a hydrophobic moietysubstituted for, or appended to, at least one amino acid intermediate tothe N-terminal and C-terminal amino acids.
 10. The protein of claim 3,wherein the lipid moiety is a fatty acid selected from saturated andunsaturated fatty acids having between 2 and 24 carbon atoms.
 11. Theprotein of claim 1, wherein the protein is a hedgehog protein obtainablefrom a vertebrate source.
 12. The protein of claim 11, wherein thehedgehog is obtainable from a human or rat.
 13. The protein of claim 11,wherein the vertebrate hedgehog is selected from the group consisting ofSonic, Indian, and Desert hedgehog.
 14. The protein of claim 1, furthercomprising a vesicle in contact with the hydrophobic moiety.
 15. Theprotein of claim 14, wherein the vesicle is selected from the groupconsisting of a cell membrane, a micelle, and a liposome.
 16. Theprotein of claim 11, wherein the hedghog protein has an amino acidsequence according to any one of SEQ ID NOS: 1-4.
 17. The protein ofclaim 13, wherein the hedgehog protein is missing between 1 and about 10amino acids from the C-terminus thereof, when compared to a wild-typehedgehog protein.
 18. The protein of claim 16, wherein the protein hasat least 60% amino acid identity to Sonic, Indian or Desert hedgehog.19. An isolated, protein of the form: A-Cys-[Sp]-B-[Sp]-X, wherein A isa hydrophobic moiety; Cys is a cysteine or functional equivalentthereof; [Sp] is an optional spacer peptide sequence; B is a proteincomprising a plurality of amino acids and, optionally, another spacerpeptide sequence; and X is optionally another hydrophobic moiety linkedto an amino acid of protein B.
 20. The isolated protein of claim 19,wherein the isolated protein is a hedgehog protein.
 21. The isolatedprotein of claim 20, wherein, if X is present, then it is cholesterol.22. The isolated protein of claim 19, wherein protein B is modified atat least one other amino acid with at least one hydrophobic moiety. 23.The isolated protein of claim 19, wherein the A-Cys linkage is via anamino group of cysteine.
 24. The isolated protein of claim 19, furthercomprising a vesicle in contact therewith.
 25. The isolated protein ofclaim 24, wherein the vesicle in contact therewith is selected from thegroup consisting of a cell membrane, micelle and liposome.
 26. A vesicleto which is attached a plurality of molecules, at least two of theplurality having the form of claim
 19. 27. The vesicle of claim 26,wherein the vesicle is selected from the group consisting of a cellmembrane, liposome and micelle.
 28. An isolated, protein having aC-terminal amino acid and an N-terminal thioproline group, said groupformed by reacting an aldehyde with an N-terminal cysteine of theprotein.
 29. An isolated, protein having a C-terminal amino acid and anN-terminal amide group, said group formed by reacting a fatty acidthioester with an N-terminal cysteine of the protein.
 30. An isolated,protein having a C-terminal amino acid and an N-terminal maleimidegroup, said N-terminal maleimide group formed reacting a maleimide groupwith the N-terminal cysteine of the protein.
 31. The isolated protein ofclaims 28, 29 or 30, wherein the C-terminal amino acid of the protein ismodified with an hydrophobic moiety.
 32. The isolated protein of claim31, wherein the protein is a hedgehog protein.
 33. The isolated proteinof claim 32, wherein the C-terminal hydrophobic moiety is cholesterol.34. A method of generating a multivalent protein complex comprising thestep of linking, in the presence of a vesicle, a hydrophobic moiety toan N-terminal cysteine of a protein, or a functional equivalent of theN-terminal cysteine.
 35. The method of claim 34, wherein the step oflinking comprises linking a lipid moiety which is selected fromsaturated and unsaturated fatty acids having between 2 and 24 carbonatoms.
 36. The method of claim 34, wherein the protein is a hedgehogprotein.
 37. The method of claim 36, wherein the hedgehog is selectedfrom the group consisting of Sonic, Indian and Desert hedgehog.
 38. Themethod of claim 36, wherein the hedghog has an amino acid sequenceaccording to any one of SEQ ID NOS: 1-4.
 39. The method of claim 34,wherein the step of linking comprises linking with a vesicle selectedfrom the group consisting of a cell membrane, liposome and micelle. 40.A method for modifying a physico-chemical property of a protein,comprising introducing at least one hydrophobic moiety to an N-terminalcysteine of the proteinor to a functional equivalent of the N-terminalcysteine.
 41. The method of claim 40, further comprising contacting thehydrophobic moiety with a vesicle.
 42. The method of claim 40, whereinthe hydrophobic moiety is either a lipid moiety selected from saturatedand an unsaturated fatty acids having between 2 and 24 carbon atoms oris a hydrophobic protein.
 43. The method of claim 40, wherein theprotein is a hedgehog protein.
 44. The method of claim 43, wherein thehedgehog protein is selected from the group consisting of Sonic, Indianand Desert hedgehog.
 45. The method of claim 43, wherein the hedgehoghas an amino acid sequence according to any one of SEQ ID NOS: 1-4. 46.The method of claim 41, wherein the step of contacting comprisescontacting with a vesicle selected from the group consisting of a cellmembrane, liposome and micelle.
 47. A protein complex, produced by themethod of claim
 34. 48. A modified protein, produced by the method ofclaim
 40. 49. The complex of claim 47, wherein the protein is selectedfrom the group consisting of gelsolin; an interferon, an interleukin,tumor necrosis factor, monocyte colony stimulating factor, granulocytecolony stimulating factor, granulocyte macrophage colony stimulatingfactor, erythropoietin, platelet derived growth factor, growth hormoneand insulin.
 50. A method for modifying a protein having a biologicalactivity and containing an N-terminal cysteine, comprising reacting theN-terminal cysteine with a fatty acid thioester to form an amide,wherein such modification enhances the protein's biological activity.51. The method of claim 50, wherein the protein is a hedgehog protein.52. The method of claim 51, wherein the hedgehog protein is selectedfrom the group consisting of Sonic, Indian, Desert hedgehog, andfunctional variants thereof.
 53. A method for modifying a protein havinga biological activity and containing an N-terminal cysteine, comprisingreacting the N-terminal cysteine with a maleimide group, wherein suchmodification enhances the protein's biological activity.
 54. The methodof claim 53, wherein the protein is a hedgehog protein.
 55. The methodof claim 54, wherein the hedgehog protein is selected from the groupconsisting of Sonic, Indian, Desert hedgehog, and functional variantsthereof.
 56. A method for modifying a protein having a biologicalactivity comprising appending an hydrophobic peptide to the protein. 57.The method of claim 56, wherein the hydrophobic peptide is appended toan amino acid of the protein selected from the group consisting of theN-terminal amino acid, the C-terminal amino acid, an amino acidintermediate between the N-terminal amino acid and the C-terminal aminoacid, and combinations of the foregoing.
 58. The method of claim 69,wherein the protein is a hedgehog protein.
 59. The method of claim 71,wherein the hedgehog protein is selected from the group consisting ofSonic, Indian and Desert hedgehog.
 60. A therapeutic use of the proteinof any of claims 1 or 20, comprising administering the protein to asubject.
 61. A method of treating a neurological disorder in a patientcomprising administering to the patient a protein of any of claims 1 or20.
 62. The protein of claim 1, wherein the protein is an extracellularsignaling protein.
 63. The method of claim 57, wherein the step ofappending comprises replacing at least the N-terminal amino acid of theprotein with at least one hydrophobic amino acid.
 64. The method ofclaim 63, wherein the at least one hydrophobic amino acid is a pluralityof isoleucine residues.
 65. The method of claim 63, further comprisingchemically modifying at least one of the isoleucine residues.
 66. Anisolated, protein having a C-terminal amino acid and an N-terminalacetamide group, said group formed by reacting a substituted acetamidewith an N-terminal cysteine of the protein.
 67. An isolated, proteinhaving a C-terminal amino acid and an N-terminal thiomorpholine group,said group formed by reacting a haloketone group with an N-terminalcysteine of the protein.
 68. A method for modifying a protein having abiological activity and containing an N-terminal cysteine, comprisingreacting the N-terminal cysteine with a substituted acetamide group,wherein such modification enhances the protein's biological activity.69. The method of claim 68, wherein the protein is a hedgehog protein.70. The method of claim 69, wherein the hedgehog protein is selectedfrom the group consisting of Sonic, Indian, Desert hedgehog, andfunctional variants thereof.
 71. A method for modifying a protein havinga biological activity and containing an N-terminal cysteine, comprisingreacting the N-terminal cysteine with a aloketone group, wherein suchmodification enhances the protein's biological activity.
 72. The methodof claim 71, wherein the protein is a hedgehog protein.
 73. The methodof claim 72, wherein the hedgehog protein is selected from the groupconsisting of Sonic, Indian, Desert hedgehog, and functional variantsthereof.
 74. A hedgehog polypeptide modified with one or more lipophilicmoieties with the proviso that, in the instance wherein the hedgehogpolypeptide is the mature N-terminal proteolytic fragment of a hedgehogprotein, the lipophilic moiety is other than a sterol at the C-terminalresidue.
 75. A hedgehog polypeptide modified with one or more lipophilicmoieties at internal amino acid residues.
 76. A hedgehog polypeptidemodified with one or more lipophilic aromatic hydrocarbons.
 77. Thehedgehog polypeptide of any of claims 74-76 which polypeptide isprovided as a purified protein preparation.
 78. The hedgehog polypeptideof any of claims 74-76 which polypeptide is provided as a pharmaceuticalpreparation.
 79. The hedgehog polypeptide of claim 74 or 75, wherein thelipophilic moieties are selected from the group consisting of fattyacids, lipids, esters, alcohols, cage structures, and aromatichydrocarbons.
 80. The hedgehog polypeptide of claim 76 or 79, whereinthe aromatic hydrocarbon is selected from the group consisting ofbenzene, perylene, phenanthrene, anthracene, naphthalene, pyrene,chrysene, and naphthacene.
 81. The hedgehog polypeptide of claim 80,wherein the aromatic hydrocarbon is a pyrene.
 82. The hedgehogpolypeptide of claim 74 or 75, wherein the lipophilic moieties areselected from the group consisting of isoprenoids, terpenes andpolyalicyclic hydrocarbons.
 83. The hedgehog polypeptide of claim 82,wherein the lipophilic moieties are selected from the group consistingof adamantanes, buckminsterfullerenes, vitamins, polyethylene glycol,oligoethylene glycol, (C1-C18)-alkyl phosphate diesters,—O—CH2—CH(OH)—O—(C12-C18)-alkyl.
 84. The hedgehog polypeptide of claim83, wherein the lipophilic moieties are selected from the groupconsisting of 1- or 2-adamantylacetyl, 3-methyladamant-1-ylacetyl,3-methyl-3-bromo-1-adamantylacetyl, 1-decalinacetyl, camphoracetyl,camphaneacetyl, noradamantylacetyl, norbornaneacetyl,bicyclo[2.2.2.]-oct-5-eneacetyl,1-methoxybicyclo[2.2.2.]-oct-5-ene-2-carbonyl,cis-5-norbornene-endo-2,3-dicarbonyl, 5-norbornen-2-ylacetyl,(1R)-(-)-myrtentaneacetyl, 2-norbornaneacetyl,anti-3-oxo-tricyclo[2.2.1.0<2,6>]-heptane-7-carbonyl, decanoyl,dodecanoyl, dodecenoyl, tetradecadienoyl, decynoyl and dodecynoyl. 85.The hedgehog polypeptide of any of claims 74-76, wherein the lipophilicmoiety or moieties potentiate the biological activity of the polypeptiderelative to the modified hedgehog polypeptide.
 86. A method for alteringthe growth state of a cell responsive to hedgehog signaling, comprisingcontacting the cell with a lipophilic-modified hedgehog polypeptide ofany of claims 74-76.